Materials and methods relating to protein aggregation in neurodegenerative disease

ABSTRACT

The present invention provides methods of proteolytically converting a precursor protein (e.g. tau) to a product fragment (e.g., a 12 kd fragment) in a stable cell line, wherein the precursor protein is associated with a disease state in which the precursor protein aggregates pathologically (e.g. a tauopathy), and the methods comprise: (a) providing a stable cell line transfected with nucleic acid encoding: (i) a template fragment of the precursor protein such that the template fragment is constitutively expressed in the cell at a level which is not toxic to the cell; and (ii) the precursor protein, which protein is inducibly expressed in the cell in response to a stimulus, whereby interaction of the template fragment with the precursor protein causes a conformational change in the precursor protein such as to cause aggregation and proteolytic processing of the precursor protein to the product fragment. The method is preferably used to screen for modulators of the aggregation process by monitoring production (or modulation of production) of the product band or bands. Also provided are materials for used in the assays, plus medicaments, and related uses and processes, based on compounds which show high activity in the assay of the invention e.g. reduced diaminophenothiazines.

This application is the National Phase Application of InternationalApplication PCT/GB02/00153 filed on Jan. 15, 2002.

TECHNICAL FIELD

The present invention concerns cell-based models and other test systemsfor modelling the aggregation of proteins associated withneurodegenerative disease. It further relates to compounds capable ofmodulating such aggregation.

BACKGROUND ART

Conditions of dementia such as Alzheimer's disease (AD) are frequentlycharacterised by a progressive accumulation of intracellular and/orextracellular deposits of proteinaceous structures such as β-amyloidplaques and neurofibrillary tangles in the brains of affected patients.The appearance of these lesions largely correlates with pathologicalneurofibrillary degeneration and brain atrophy, as well as withcognitive impairment (Mukaetova-Ladinska, E. B. et al. (2000) Am. J.Pathol. Vol. 157, No. 2, 623-636).

Both neuritic plaques and neurofibrillary tangles contain paired helicalfilaments (PHFs), of which a major constituent is themicrotubule-associated protein tau (Wischik et al. (1988) PNAS USA 85,4506). Plaques also contain extracellular β-amyloid fibrils derived fromthe abnormal processing of amyloid precursor protein (APP; Kang et al.(1987) Nature 325, 733). An article by Wischik et al. (in ‘Neurobiologyof Alzheimer's Disease’, 2nd Edition (2000) Eds. Dawbarn, D. and Allen,S. J., The Molecular and Cellular Neurobiology Series, Bios ScientificPublishers, Oxford) discusses in detail the putative role of tau proteinin the pathogenesis of neurodegenerative dementias.

Studies of Alzheimer's disease indicate that the loss of the normal formof tau (Mukaetova-Ladinska et al. (1993) Am. J. Pathol., 143, 565;Wischik et al. (1995a) Neurobiol. Ageing, 16: 409; Lai et al. (1995b)Neurobiol. Ageing, 16: 433), accumulation of pathological PHFs(Mukaetova-Ladinska et al. (1993), loc. cit.; Harrington et al. (1994a)Dementia, 5, 215; Harrington et al. (1994b) Am. J. Pathol., 145, 1472;Wischik et al., (1995a), loc. cit.) and loss of synapses in themid-frontal cortex (Terry et al. (1991) Ann. Neurol., 30, 572) correlatewith associated cognitive impairment. Furthermore, loss of synapses(Terry et al., loc. cit.) and loss of pyramidal cells (Bondareff et al.(1993) Arch. Gen. Psychiatry, 50: 350) both correlate with morphometricmeasures of tau-reactive neurofibrillary pathology, which parallels, ata molecular level, an almost total redistribution of the tau proteinpool from a soluble to a polymerised form (PHFs) in Alzheimer's disease(Mukaetova-Ladinska et al. (1993), loc. cit.; Lai et al. (1995), loc.cit.).

Tau exists in alternatively-spliced isoforms, which contain three orfour copies of a repeat sequence corresponding to themicrotubule-binding domain (Goedert, M., et al. (1989) EMBO J. 8,393-399; Goedert, M., et al. (1989) Neuron 3, 519-526). Tau in PHFs isproteolytically processed to a core domain (Wischik, C. M., et al.(1988) Proc. Natl. Acad. Sci. USA 85, 4884-4888; Wischik et al. PNAS USA1988, 85:4506-4510); Novak, M., et al. (1993) EMBO J. 12, 365-370) whichis composed of a phase-shifted version of the repeat domain; only threerepeats are involved in the stable tau-tau interaction (Jakes, R., etal. (1991) EMBO J. 10, 2725-2729) Once formed, PHF-like tau aggregatesact as seeds for the further capture and provide a template forproteolytic processing of full-length tau protein (Wischik et al. 1996Proc Natl Acad Sci USA 93, 11213-11218).

In the course of their formation and accumulation, paired helicalfilaments (PHFs) first assemble to form amorphous aggregates within thecytoplasm, probably from early tau oligomers which become truncatedprior to, or in the course of, PHF assembly (Mena, R., et al. (1995)Acta Neuropathol. 89, 50-56; Mena, R., et al. (1996) Acta Neuropathol.91, 633-641). These filaments then go on to form classical intracellularneurofibrillary tangles. In this state, the PHFs consist of a core oftruncated tau and a fuzzy outer coat containing full-length tau(Wischik., C. M., et al, (1996) loc. cit.). The assembly process isexponential, consuming the cellular pool of normal functional tau andinducing new tau synthesis to make up the deficit (Lai, R. Y. K., etal., (1995), Neurobiology of Ageing, Vol. 16, No. 3, 433-445).Eventually, functional impairment of the neurone progresses to the pointof cell death, leaving behind an extracellular tangle. Cell death ishighly correlated with the number of extracellular tangles (Wischik etal. 2000, loc.cit). As tangles are extruded into the extracellularspace, there is progressive loss of the fuzzy outer coat of theneurone-PHF with corresponding loss of N-terminal tau immunoreactivity,but preservation of tau immunoreactivity associated with the PHF core(FIG. 1; also Bondareff, W. et al., (1994) J. Neuropath. Exper. Neurol.,Vol. 53, No. 2, 158-164).

The phase shift which is observed in the repeat domain of tauincorporated into PHFs suggests that the repeat domain undergoes aninduced conformational change during incorporation into the filament.During the onset of Alzheimer's disease, it is envisaged that thisconformational change could be initiated by the binding of tau to apathological substrate, such as damaged or mutated membrane proteins(see FIG. 2—also Wischik, C. M., et al. (1997) in“Microtubule-associated proteins: modifications in disease”, eds. Avila,J., Brandt, R. and Kosik, K. S. (Harwood Academic Publishers, Amsterdam)pp.185-241).

In the case of Alzheimer's disease, current pharmaceutical therapies arefocused on symptomatic treatment of the loss of cholinergic transmissionwhich results from neurodegeneration (Mayeux, R., et al. (1999) New Eng.J. Med. 341, 1670-1679). However, although the available treatmentsdelay progression of the disease for up to six to twelve months, they donot prevent it. The discovery of drugs that could prevent theaggregation of tau which leads to neurodegeneration would provide a moreeffective strategy for prophylaxis or for inhibiting the progression ofthe disease, which would not require an immediate knowledge of thediverse upstream events that initiate the aggregation (see FIG. 3).

Models and Assays

WO 96/30766 describes an in vitro assay for tau aggregation in which afragment of tau corresponding to the core repeat domain, which has beenadsorbed to a solid phase substrate, is able to capture solublefull-length tau and bind tau with high affinity (see FIG. 4). Thisassociation confers stability against digestion of proteases on the taumolecules on the repeat domains of tau molecules which have aggregated.The process is self-propagating, and can be blocked selectively byprototype pharmaceutical agents ((Wischik et al. 1996 Proc Natl Acad SciUSA 93, 11213-11218).

Although the in vitro assay described in WO 96/30766 enables theidentification of inhibitors or modulators of tau-tau association, thepresent inventors have also recognized that cell-based models ofAlzheimer's disease-like protein aggregation would be useful. Suchcellular models could be used both in the primary screening of candidatemodulators of tau-tau aggregation, and in the secondary screening ofcompounds already identified in the in vitro assay of WO 96/30766.Furthermore, the demonstration of tau aggregation in cells could alsoaid in the identification of normal cellular substrates which areinvolved in the initiation of pathological tau aggregation, whichsubstrates could themselves be targets for pharmaceutical intervention.

However, numerous papers reporting the expression of various tauconstructs in tissue culture models have failed to demonstrateaggregation (see e.g. Baum, L. et al., (1995) Mol. Brain Res. 34:1-17).For instance, 3T3 mouse fibroblasts do not possess tau protein and thuspresent a cellular environment in which recombinant tau can be expressedindependent of endogenous mouse tau. Transfection of various cell lineshas been reported previously (Kanai et al., 1989; Goedert and Jakes,1990; Knops et al, 1991; Lee and Rook, 1992; Gallo et al., 1992; Lo etal., 1993; Montejo de Garcini et al., 1994; Fasulo et al., 1996).However the stable long term expression of truncated tau in such celllines was not achieved. For example, tau constructs for residues 164 or173 to 338 or 352 did not express protein (Lee and Rook, 1992).

Although Fasulo et al. (Alzheimer's Research 1996, 2, 195-200) reportedtransient expression of truncated tau in COS cells, data for stable longterm expression of this tau was not shown. These workers concluded fromthe use of the transient transfection system that expression oftruncated tau by itself was not sufficient to induce tau aggregation ina manner suitable for testing drugs.

Thus far, the aggregation of soluble tau in vitro has only been achievedunder non-physiological conditions and at high concentrations (reviewedin Wischik (2000), loc. cit).

WO 96/30766 describes two approaches for studying tau aggregation in acellular environment. In the first approach, full-length tau orfragments of tau were stably expressed in cells. In the second approach,aggregated tau was transiently transfected into cells by use oflipofectin.

Although both of these approaches are useful for the study of tau-tauaggregation, they have some limitations. Transfection of aggregated tauinto cells using lipofection is of variable efficiency, as is theproduction in vitro of aggregated tau itself. Moreover, the core taufragment, which is the most efficient seed for tau aggregation, is foundto be toxic when stably expressed in cells, leading to low expressionlevels. Thus, constitutive expression of the truncated tau fragment ofthe PHF core in eukaryotic cells is difficult to achieve. Transientexpression systems permit the optimization of expression of tau, but theinherent toxicity of the fragments renders even these systemsunreliable. Longer fragments of tau are less toxic, but these do notreliably aggregate when expressed in cells.

Thus it would be desirable for an alternative model system to bedeveloped, in which the interaction between e.g. tau molecules and thelike could be investigated under physiological conditions, in a stableand controllable cell line, and which could be used to screen forpotential diagnostic, prognostic or therapeutic agents of conditionssuch as Alzheimer's disease.

DISCLOSURE OF THE INVENTION

The present inventors have devised a stable cellular test system whichcan be used to model the template-driven proteolytic processing of aprotein, the aggregation of which is associated with neurodegenerativedisease. In one test system, exemplified with the tau protein, very lowlevel constitutive expression of a fragment of the tau protein wascombined with inducible expression of full-length tau. Induction of thefull-length tau lead to its proteolytic conversion to a processedfragment, confirming that “templated proteolytic processing” of the tauwas occurring. The system readily permits the demonstration of theeffects of tau aggregation inhibitors through their inhibition ofproduction of the processed, 12 kD, fragment from induced full-lengthtau.

That such a stable system can be achieved notwithstanding the inherenttoxic properties of the 12 kD fragment is particularly surprising. Forinstance, as demonstrated in the Examples below, although partialtruncation at either N- or C-termini of full-length tau results in celllines in which stable expression can be maintained, these longerconstructs show only a weak propensity to aggregate, rather than bindingto the microtubular network. Stable expression of combinations of taufragments generates aggregates within the cytoplasm of cells, but thissystem cannot be maintained reproducibly. Systems based on the inducibleexpression of the 12 kD fragment lead to toxicity as a result ofunpredictable intracellular aggregation of the fragment.

Thus there would appear to be a trade-off in stable expression cellsystems between inducing aggregation and hence toxicity on the one hand,which produces cell lines which are either variable or non-viable, andmaintaining viable cell lines in which tau has a low propensity toaggregate. Notwithstanding this, the inducible tau expression system ofthe present invention is both stable, and yet able to provide controlledaggregation of protein for use in screens and the like.

Additionally, use of the assay has provided evidence that the mechanismof action of certain inhibitors (e.g. phenothiazines) of proteinaggregation is primarily steric in nature, rather than essentiallyredox, as may have been suspected on the basis of the prior art. Thisdiscovery has unexpected implications for the choice, assessment,formulation and use of such compounds in the context of the diseasesdiscussed herein. In particular, it shows that assessment of diffusioncoefficients can provide a valuable screen for identifying putativeinhibitors, or optimising the structure or state of known ones, becausethe parameters inherently assessed by measuring the diffusioncoefficient may be highly relevant to the inhibitors' potency.

The assay further shows that use of phenothiazines in their reduced formcan be advantageous for enhancing their inhibitory properties. Theseobservations form the basis of further aspects of the present invention.

In general the present invention provides a method for converting,through proteolytic processing, a precursor protein to a productfragment of the precursor protein, in a stable cell line, which methodcomprises the steps of: (a) providing a stable cell line transfectedwith nucleic acid encoding (i) a template fragment of the precursorprotein such that the template fragment is constitutively expressed inthe cell at a level which is not toxic to the cell; and (ii) theprecursor protein, which protein is inducibly expressed in the cell inresponse to a stimulus, whereby interaction of the template fragmentwith the precursor protein causes a conformational change in theprecursor protein such as to cause aggregation and proteolyticprocessing of the precursor protein to the product fragment.

The method may include subjecting the cell to the stimulus such that theprecursor protein is expressed in the cell. However in embodiments wherean inducible promoter is used which causes low, but detectable levels ofexpression even in the absence of the stimulus, then the stimulus stepmay be omitted.

Generally speaking, the precursor protein will be one which, in vivo, iscapable of undergoing an induced conformational polymerisationinteraction (in a self-propagating manner) leading ultimately to theformation of aggregates comprised of the product fragment, andassociated with the disease state. The product fragment obtained in themethod provided herein is a measure of the pathological aggregation andproteolysis process which in vivo leads to the production of one or moretoxic products and the disease state. The product fragment (or one ormore of the fragments) of the present method may be toxic, or may simplybe used as an indicator of the pathological aggregation process.

The proteins and interactions upon which the method is based arediscussed in more detail below.

The present inventors have demonstrated that it is unexpectedly possibleto constitutively express the template fragment at a (first)concentration which is not toxic to the cell line i.e. the cell line isviable. Nor does it show cellular abnormalities of the sort shown e.g.in WO 96/30766 at FIG. 29.

Nevertheless (e.g. at a time predetermined by addition of the stimulus)it is possible to seed the processing of the precursor protein to aproduct fragment (which may be the same, broadly equivalent, or quitedifferent to the template fragment) which can thus accumulate to a(second, higher) concentration which is toxic to the cell and whichcorresponds to the disease state. This in turn provides convenientmethods for modeling the disease state associated with the effects ofthe product fragment, and assessing the effect of modulators on thegeneration of the product fragment.

In various other, discrete, embodiments the invention providescorresponding methods for any of initiating, seeding, or controlling theproteolytic processing and optionally aggregation of the precursorprotein to the product fragment.

In each case the method may involve monitoring (directly or indirectly)the level of proteolytic processing of the precursor protein.

In one embodiment of the present invention fibroblast cells (3T6)express full-length tau (“T40”) under the control of an induciblepromotor and low constitutive levels of the PHF-core tau fragment (12 kDfragment). When T40 expression is induced in this system, it undergoesaggregation-dependent truncation within the cell, N-terminally at˜a.a.295 and C-terminally at ˜a.a.390, thereby producing higher levelsof the 12 kD PHF-core domain fragment.

Production of the 12 kD fragment can be blocked in a dose-dependentmanner by tau-aggregation inhibitors. Indeed the quantitation ofinhibitory activity of compounds with respect to proteolytic generationof the 12 kD fragment within cells can be described entirely in terms ofthe same parameters which describe inhibition of tau-tau binding invitro. That is, extent of proteolytic generation of the 12 kD fragmentwithin cells is determined entirely by the extent to tau-tau bindingthrough the repeat domain. The availability of the relevant proteaseswithin the cell is non-limiting.

Precursor Proteins and Diseases (Including Tauopathies)

As stated above, the invention may be based around the use of anyprotein which is associated with a disease in which the proteinundergoes an induced conformational polymerisation interaction i.e. onein which a conformational change of the protein, or in a fragmentthereof, gives rise to templated binding and aggregation of further(precursor) protein molecules in a self-propagating manner.

Once nucleation is initiated, an aggregation cascade may ensue whichinvolves the induced conformational polymerisation of further proteinmolecules, leading to the formation of toxic product fragments inaggregates which are substantially resistant to further proteolysis. Theprotein aggregates thus formed are thought to be a proximal cause ofneurodegeneration, clinical dementia, and other pathological symptoms ofthis group of diseases.

Preferred embodiments of the invention are based on tau protein. Whereused herein, the term “tau protein” refers generally to any protein ofthe tau protein family. Tau proteins are characterised as being oneamong a larger number of protein families which co-purify withmicrotubules during repeated cycles of assembly and disassembly(Shelanski et al. (1973) Proc. Natl. Acad. Sci. USA, 70., 765-768), andare known as microtubule-associated-proteins (MAPs). Members of the taufamily share the common features of having a characteristic N-terminalsegment, sequences of approximately 50 amino acids inserted in theN-terminal segment, which are developmentally regulated in the brain, acharacteristic tandem repeat region consisting of 3 or 4 tandem repeatsof 31-32 amino acids, and a C-terminal tail.

MAP2 is the predominant microtubule-associated protein in thesomatodendritic compartment (Matus, A., in “Microtubules” [Hyams andLloyd, eds.] pp 155-166, John Wiley and Sons, NY). MAP2 isoforms arealmost identical to tau protein in the tandem repeat region, but differsubstantially both in the sequence and extent of the N-terminal domain(Kindler and Garner (1994) Mol. Brain Res. 26, 218-224). Nevertheless,aggregation in the tandem-repeat region is not selective for the taurepeat domain. Thus it will be appreciated that any discussion herein inrelation to tau protein or tau-tau aggregation should be taken asrelating also to tau-MAP2 aggregation, MAP2-MAP2 aggregation and so on.

FIG. 5 shows a Table listing various other disease-associatedaggregating proteins which may be used in the present invention. In eachcase the disease or diseases in which the initiation of aggregationand\or mutation of the protein(s) may play a role is also listed. Thedomain or mutation responsible for the disease activity is listed, andat least all or part of this minimal portion of the protein wouldpreferably be encompassed by the template fragment used in the presentinvention.

As can be seen from the table, example diseases which are characterisedby pathological protein aggregation include motor neurone disease andLewy body disease.

Notably it is not only Alzheimer's Disease in which tau protein (andaberrant function or processing thereof) may play a role. Thepathogenesis of neurodegenerative disorders such as Pick's disease andProgressive Supranuclear Palsy (PSP) appears to correlate with anaccumulation of pathological truncated tau aggregates in the dentategyrus and stellate pyramidal cells of the neocortex, respectively. Otherdementias include fronto-temporal dementia (FTD); parkinsonism linked tochromosome 17 (FTDP-17); disinhibition-dementia-parkinsonism-amyotrophycomplex (DDPAC); pallido-ponto-nigral degeneration (PPND); Guam-ALSsyndrome; pallido-nigro-luysian degeneration (PNLD); cortico-basaldegeneration (CBD) and others (see Wischik et al. 2000, loc. cit, fordetailed discussion—especially Table 5.1). All of these diseases, whichare characterized primarily or partially by abnormal tau aggregation,are referred to herein as “tauopathies”.

Thus it will be appreciated, in the light of the above discussion, (andexcept where context requires otherwise) where the embodiments of theinvention are described with respect to tau protein or tau-like proteins(e.g. MAP2) the description should be taken as applying equally to theother proteins discussed above (e.g. β-amyloid, synuclein, prion etc.)or other proteins which may initiate or undergo a similar pathologicalaggregation by virtue of conformational change in a domain critical forpropagation of the aggregation, or which imparts proteolytic stabilityto the aggregate this formed (article by Wischik et al. (in“Neurobiology of Alzheimer's Disease”, 2nd Edition (2000) Eds. Dawbarn,D. and Allen, S. J., The Molecular and Cellular Neurobiology Series,Bios Scientific Publishers, Oxford). All such proteins may be referredto herein as “aggregating disease proteins.”

Likewise, where mention is made herein of “tau-tau aggregation”, or thelike, this may also be taken to be applicable to other“aggregating-protein aggregation”, such as β-amyloid aggregation, prionaggregation and synuclein aggregation etc. Likewise “tau proteolyticdegradation” and so on.

Template Fragments

In preferred embodiments of the present invention, the templatefragment, comprises, consists essentially of, or consists of a “corefragment” of the precursor protein, which term refers to that part ofthe protein that is able to bind to the precursor protein to initiate orpropagate proteolysis and aggregation as described above.

In the case of disease proteins which aggregate, such core fragments arealso likely to be those which contribute to the proteolytic stability ofthe aggregate.

Thus, for example, a “tau core fragment” is a tau fragment comprising atruncated tau protein sequence derived from the tandem repeat regionand, which, in the appropriate conditions, is capable of binding to thetandem repeat region of a further tau protein or a MAP2 protein withhigh affinity. In the case of tau, the preferred fragment is thusexemplified by, but not limited to, the tau fragments present in PHFs(and, ultimately, neurofibrillary tangles) in Alzheimer's diseasebrains.

A preferred tau fragment may thus be from about (say) between 295-297extending to about 390-391 (see ‘dGAE’ in FIG. 6) although variants ofsuch fragments may also be used, as discussed below.

In the case of APP (amyloid precursor protein), for instance, expressionof a fragment of the APP that encompasses the Aβ domain of 1-40 or 1-42amino acids as a fusion protein, may be preferred.

Other core fragments may be based e.g. on the domains discussed withreference to FIG. 5. Template fragments may include domains from two, ormore than two, of these proteins (e.g. as fusions). The total length ofthe template fragment may be any which is appropriate to the assay andaggregation disease protein core fragment being used, but will generallybe greater than or equal to about 20, 30, 40, 50, 60, 70, 80, 90, or soamino acids in length. However in some embodiments it may be greaterthan 100, 200 or even 500, if this is desired.

Derivatives

In all instances herein where a named protein (e.g. precursor protein,template or core fragment) or a recited nucleic acid sequence isdiscussed, a derivative or other variant of the corresponding referenceprotein (or nucleic acid) may be used as appropriate, provided that itretains appropriate characteristics of the reference sequence. Suchderivatives will also share sequence identity with the referencesequence.

For instance the protein used may include an extended N- or C-terminus,which extension may be heterologous to the protein sequence. Equally,the derivative will be one by way of amino acid insertion, deletion, oraddition of the reference sequence. For example, a tau protein, or taucore fragment, derivative will comprise at least a partial amino acidsequence resembling the tandem repeat region of the tau proteins, but inwhich one or more of the amino acids of the natural tau or its fragmentshave been replaced or deleted, or into which other amino acids have beeninserted.

Such changes may be made to enhance or ablate binding activity (thelatter case being useful for control experiments). Controls may containdeletions of sequences or domains to see what effect on aggregationthese may have.

Preferred derivatives may be those which incorporate mutationscorresponding to those known or suspected to be associated with thedisease state. These may include changes corresponding to P301S withinthe tau sequence (see FIG. 7). Other mutations include G272V, G389R,P301L, N279K, S305N, V337M, G272V, K280Δ, R406w (see also Wischik et al,2000, supra).

Other preferred derivatives may include tandem repeats of thecore-fragments discussed above, or binding domains within thosefragments.

Yet further derivatives may be based on chimeric products based onmultiple, related, disease proteins in which their sequences are mixedor combined. For example restriction enzyme fragments of tau could beligated together with fragments of MAP2 or even of an unrelated gene togenerate recombinant derivatives. An alternative strategy for modifyingthe core fragments would employ PCR as described by Ho et al., 1989,Gene 77, 51-59 or DNA shuffling (Crameri et al., 1998 Nature 391).

Use of Nucleic Acid Constructs

Nucleic acids of, or for use in, the present invention may be providedisolated and/or purified from their natural environment, insubstantially pure or homogeneous form, or free or substantially free ofother nucleic acids of the species of origin. Where used herein, theterm “isolated” encompasses all of these possibilities. Nucleic acidse.g. encoding the template fragment, will be at least partiallysynthetic in that it will comprise nucleic acid sequences which are notfound together in nature (do not run contiguously) but which have beenligated or otherwise combined artificially.

Nucleic acid according to the present invention may be in the form of,or derived from, cDNA, RNA, genomic DNA and modified nucleic acids ornucleic acid analogs. Where a DNA sequence is specified, e.g. withreference to a figure, unless context requires otherwise the RNAequivalent, with U substituted for T where it occurs, is encompassed.

As described above, the nucleic acids may encode derivatives or othervariants sharing homology with the reference sequences in question.Preferably, the nucleic acid and/or amino acid sequence in questionwould share about 50%, or 60%, or 70%, or 80% identity, most preferablyat least about 90%, 95%, 96%, 97%, 98% or 99% of the sequence upon whichthe variant is based. Similarity or homology may be as defined anddetermined by the TBLASTN program, of Altschul et al. (1990) J. Mol.Biol. 215: 403-10, which is in standard use in the art, or, and this maybe preferred, the standard program BestFit, which is part of theWisconsin Package, Version 8, September 1994, (Genetics Computer Group,575 Science Drive, Madison, Wis., USA, Wisconsin 53711) using thedefault parameters. One common formula for calculating the stringencyconditions required to achieve hybridization between nucleic acidmolecules of a specified sequence homology is: T_(m)=81.5° C.+16.6 Log[Na+]+0.41 (% G+C)−0.63 (% formamide)−600/#bp in duplex.

Nucleic acid sequences which encode the appropriate proteins orpolypeptides can be readily prepared by the skilled person using theinformation and references contained herein and techniques known in theart (for example, see Sambrook, Fritsch and Maniatis, “MolecularCloning, A Laboratory Manual”, Cold Spring Harbor Laboratory Press,1989, and Ausubel et al., Short Protocols in Molecular Biology, JohnWiley and Sons, 1992). These techniques include (i) the use of thepolymerase chain reaction (PCR) to amplify samples of the relevantnucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or(iii) preparation of cDNA sequences.

DNA encoding e.g. tau core fragments may be generated and used in anysuitable way known to those of skilled in the art, including by takingencoding DNA, identifying suitable restriction enzyme recognition siteseither side of the portion to be expressed, and cutting out said portionfrom the DNA. Modifications to the protein (e.g. tau)-encoding sequencescan be made, e.g. using site directed mutagenesis.

Constructs

Thus the invention also relates, in a further aspect, to nucleic acidmolecules encoding the appropriate precursor and template fragmentproteins. As discussed below, these may be present on the same ordifferent constructs, and in the latter case, compositions comprisingtwo or more types of construct are also provided.

Nucleic acid sequences which enable a vector to replicate in one or moreselected host cells are well known for a variety of bacteria, yeast, andviruses. For Example, various viral origins (SV40, polyoma, adenovirus,VSV or BPV) are useful for cloning vectors in mammalian cells.Expression vectors comprising a nucleic acid as described herein may,for example, be in the form of a plasmid, cosmid, viral particle, phage,or any other suitable vector or construct which can be taken up by acell and expressed appropriately.

Expression vectors will contain a promoter which is operably linked tothe protein-encoding nucleic acid sequence of interest, so as to directmRNA synthesis. Promoters recognized by a variety of potential hostcells are well known. “Operably linked” means joined as part of the samenucleic acid molecule, suitably positioned and oriented fortranscription to be initiated from the promoter. DNA operably linked toa promoter is “under transcriptional control” of the promoter.Transcription from vectors in mammalian host cells is controlled, forexample, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g. the actin promoter or an immunoglobulinpromoter, and from heat-shock promoters, provided such promoters, arecompatible with the host cell systems. Expression vectors used ineukaryotic host cells (yeast, fungi, insect, plant, animal, human, ornucleated cells from other multicellular organisms) will also containsequences necessary for the termination of transcription and forstabilizing the mRNA.

The promoter used for the template fragment will be “constitutive”. Thispromoter may be sufficiently weak that the level of template fragmentexpressed in the cell is not itself (directly) detectable usingconventional techniques, other than (indirectly) by its affect onprecursor protein, leading to aggregation and proteolytic processingthereof (i.e. effectively undetectable when said aggregation isinhibited). Such promoters may be selected by those skilled in the artin the light of the present disclosure without undue burden such asthose listed above.

In the case of the precursor protein, the promoter is “inducible”-whichis to say, and as is well understood by those skilled in the art,expression is “switched on” or increased in response to an appliedstimulus. The nature of the stimulus varies between promoters. Someinducible promoters cause little or undetectable levels of expression(or no expression) in the absence of the appropriate stimulus. Otherinducible promoters cause detectable constitutive expression in theabsence of the stimulus. Whatever the level of expression is in theabsence of the stimulus, expression from any inducible promoter isincreased in the presence of the correct stimulus. In experiments below,a Lac inducible promoter has been used.

Expression vectors of the invention may also contain one or moreselection genes. Typical selection genes encode proteins that (a) conferresistance to antibiotics or other toxins e.g. ampicillin, neomycin,methotrexate, or tetracycline, (b) complement auxotrophic deficiencies,or (c) supply critical nutrients not available from complex media, e.g.,the gene encoding D-alanine racemase for Bacilli. An example of suitableselectable markers for mammalian cells are those that enable theidentification of cells competent to take up the desiredprotein-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell, when wild-type DHFR is employed, is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad Sci USA 77:4216 (1980). A suitableselection gene for use in yeast is the trpl gene present in the yeastplasmid Rp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trplgene provides a selection marker for a mutant strain of yeast whichlacks the ability to grow in tryptophan, for example, ATCC: No. 44076 orPEP4-1 [Jones, Genetics, 85:12 (1977)].

Thus a typical vector for use in the present invention may include anorigin of replication, one or more protein sequence(s) operably linkedto a constitutive or inducible promoter as appropriate, a transcriptiontermination sequence, an enhancer element, a marker gene. Constructionof suitable vectors containing various of these components employsstandard ligation techniques which are known to the skilled artisan.

Transformation

Also provided by the present invention is a process for producing astable cell for use in a method as described above, which processcomprises the steps of: (a) introducing into a cell nucleic acidencoding (i) a template fragment of the precursor protein such that thetemplate fragment is constitutively expressed in the cell at a levelwhich is not toxic to the cell; and (ii) the precursor protein such thatthe disease protein is inducibly expressed in the cell in response to astimulus.

The introduction, which may be generally referred to without limitationas “transformation”, may employ any available technique. For eukaryoticcells, suitable techniques may include calcium phosphate transfection,DEAE-Dextran, electroporation, liposome-mediated transfection andtransduction using retrovirus or other virus, e.g. vaccinia or, forinsect cells, baculovirus. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes or other cells that contain substantialcell-wall barriers. Infection with Agrobacterium tumefaciens is used fortransformation of certain plant cells, as described by Shaw et al.,Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g, polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527 537 (1990) and Mansour et al.,Nature 336:348-352 (1988).

Host Cells

Suitable host cells for use in the invention may include bacteria,eukaryotic cells such as mammalian and yeast cells, and baculovirussystems.

Mammalian cell lines available in the art for expression of aheterologous polypeptide include fibroblast 3T6 cells, HeLa cells, babyhamster kidney cells, COS cells, monkey kidney CV1 line transformed bySV40 (COS-7, ATCC CRL 1651), Chinese hamster ovary cells/-DHFR(CHO,Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mousesertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); human lungcells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mousemammary tumour cells (MMT 060562, ATCC CCL51); and many others.

Suitable prokaryotic hosts include but are not limited to eubacteria,such as Gram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Eukaryotic microbes such as filamentous fungi or yeast are alsosuitable cloning or expression hosts for vectors. Saccharomycescerevisiae is a commonly used lower eukaryotic host microorganism. Theselection of the appropriate host cell is deemed to be within the skillin the art.

In a further aspect, the present invention provides a host cellcontaining heterologous nucleic acid of the invention as describedabove. The nucleic acid of the invention may be integrated into thegenome (e.g. chromosome) of the host cell. Integration may be promotedby inclusion of sequences which promote recombination with the genome,in accordance with standard techniques. Alternatively, the nucleic acidmay be on an extrachromosomal vector within the cell, or otherwiseidentifiably heterologous or foreign to the cell.

The cell may be produced by a method described above (introduction ofnucleic acid construct) or be the ancestor of such a cell. Correspondingcell-lines are also provided. Preferred cell-lines may be based on thefibroblast cell line, e.g. 3T6.

Host cells transfected or transformed with expression or cloning vectorsdescribed herein may be cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. The cultureconditions, such as media, temperature, pH and the like, can be selectedby the skilled artisan without undue experimentation. In general,principles, protocols, and practical techniques for maximizing theproductivity of cell cultures can be found in “Mammalian CellBiotechnology: a Practical Approach”, M. Butler, ed. JRL Press, (1991)and Sambrook et al, supra.

Gene expression can be confirmed in a sample directly, for example, byconventional Southern blotting, Northern blotting to quantitate thetranscription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205(1980)], dot blotting (DNA analysis), or in situ hybridization, using anappropriately labeled probe, based on the sequence of the aggregatingdisease protein. Alternatively, antibodies may be employed that canrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes.

Gene expression, alternatively, may be measured by immunological methodssuch as immunohistochemical staining of cells or tissue sections, andassay of cell culture, to quantitate directly the expression of geneproduct. Antibodies useful for immunohistochemical staining and/or assayof sample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal. Conveniently, the antibodies may be preparedagainst a native sequence of the aggregating disease polypeptide.

Thus one aspect of the present invention entails causing or allowingexpression from the nucleic acids discussed herein, e.g. by culturinghost cells under conditions for expression of the gene (presence ofstimulus) so that the product fragment is produced. The presentinvention also encompasses a method of producing the product fragment,the method including expression from nucleic acid as described above.

Another aspect of the present invention is a kit comprising atransformed cell or cell line as described herein, plus at least onefurther component e.g. an agent for stimulating production of theprecursor protein, or an agent for detecting the interaction of theprecursor protein with the template fragment, as described in thefollowing section.

Detection of Aggregation and\or Proteolytic Processing and\or ToxicFragment

In various embodiments, the progress of proteolytic processing oraggregation (or modulation thereof—see below) may be detected directlyor indirectly by monitoring the concentration or level any one or moreof the following species: the precursor protein; the product fragment;any by-product fragments formed during the process; an aggregate of anyof these (e.g. based on sedimentation coefficients).

Thus, as exemplified with particular tau proteins and fragments (basedon 297-351 fragment and T40), aggregation can be monitored on the basisof increasing levels of a 12 kDa processed species, derived primarilyfrom the precursor protein.

Some protein detection methods are discussed in relation to geneexpression above. Where antibodies or fragments thereof are used inembodiments of the method of the present invention may be produced byconventional techniques. Polyclonal antibodies may raised e.g. byinjecting the corresponding tau antigen into an animal, preferably arabbit, and recovering the antiserum by immunoaffinity purification, inwhich the polyclonal antibody is passed over a column to which theantigen is bound and is then eluted in a conventional manner. Preferablythe invention will use monoclonal antibodies which are selective to tauepitopes may be prepared by the method of Kohler and Milstein. Suitablemonoclonal antibodies to tau epitopes can be modified by known methodsto provide Fab fragments or (Fab′)2 fragments, chimeric, humanised orsingle chain antibody embodiments.

Antibodies according to the present invention may be modified in anumber of ways. Indeed the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimics that of an antibody enablingit to bind an antigen or epitope.

Generally speaking, where antibodies are employed for detection, theantibody may carry a reporter molecule. Alternatively, detection ofbinding may be performed by use of a second antibody capable of bindingto a first unlabelled, tau-specific antibody. In this case, the secondantibody is linked to a reporter molecule.

Antibodies may be used in any immunoassay system known in the art,including, but not limited to: radioimmunoassays, “sandwich” assays,enzyme-linked immunosorbent assays (ELISA); fluorescent immuno-assays,protein A immunoassays, etc. Typically, an immunoblot method is used.Preferably the immunoassay is performed in the solid phase, as would bewell known to the skilled person. For instance, an antibody may beadsorbed to e.g. an assay column, and the cellular sample may then bewashed through the column under conditions suitable for enabling bindingto the solid-phase antibody of any aggregate of the protein of interest,e.g. a tau-tau aggregate. Excess reagent is washed away, and the bindingof aggregated protein to the column can then be detected by any suitablemeans, e.g. as exemplified above and below.

Preferred monoclonal antibodies are as follows:

-   -   Those which recognise the N-terminal or C-terminal of the tau        epitope permit measuring of binding between truncated and        full-length tau species. Especially useful are antibodies        recognising human-specific epitopes. One such monoclonal        antibody (designated 27/499) recognises a human-specific epitope        located in the region between Gly-16 and Gln-26 of tau, and        thereby permits measurement of binding between full-length tau        species, provided one is derived from a non-human source (Lai        (1995); “The role of abnormal phosphorylation of tau protein in        the development of neurofibrillary pathology in Alzheimer's        disease”, PhD Thesis, University of Cambridge).    -   Those which recognise the core tau fragment truncated at        Glu-391. An example is mAb 423 (Novak et al. (1993), loc. cit.).        This antibody enables detection of the binding of a truncated        core tau fragment terminating at Glu-391 to a similar fragment        terminating at Ala-390, which is not recognised by mAb 423. This        truncation occurs naturally in the course of PHF assembly in        Alzheimer's disease (Mena et al. (1995), (1996), loc. cit.;        Novak et al. (1993), loc. cit.; Mena et al. (1991), loc. cit.).        Additionally, when tau is bound via the repeat domain in vitro,        digestion with a protease (e.g. pronase) generates a fragment        detectable by mAb 423 (see Wischik et al, 1996, loc cit). In the        preferred aspects of the present invention, as it relates to tau        protein, this antibody may be used to distinguish the generation        of proteolytically cleaved product fragment (Glu-391        termination) from constitutive expression of template fragment        (Ala-390).    -   Those which recognise a generic tau epitope in the repeat        domain. A preferred embodiment utilises an antibody (e.g. MAb        7.51). Where tau-MAP2 or MAP2-MAP2 aggregation is to be        detected, an antibody which detects a generic MAP2 epitope could        be used. Antibody 7.51 recognises a generic tau epitope located        in the antepenultimate repeat of tau (Novak et al. (1991) Proc.        Natl. Acad. Sci. USA, 88, 5837-5841), which is occluded when tau        is bound in a PHF-like immunochemical configuration but can be        exposed after formic acid treatment (Harrington et al. (1990),        (1991), loc. cit.; Wischik et al. (1995a), loc. cit.). Normal        soluble tau, or tau bound to microtubules, can be detected using        mAb 7.51 without formic acid treatment (Harrington et al.        (1991), loc. cit.; Wischik et al. (1995a), loc. cit.). Binding        of full-length tau in the tau-tau binding assay is associated        with partial occlusion of the mAb 7.51 epitope.

Antibody 27/342 recognises a non-species specific generic tau epitopelocated between Ser-208 and Ser-238 which is partially occluded in thecourse of the tau-tau interaction (Lai, loc. cit.).

The binding sites of some monoclonal antibodies are shown in FIG. 6.

Screening for Modulators and Inhibitors

As described above, the invention is preferably concerned with use of asystem as provided herein, in a method of modeling, and identifyingtherapeutic agents for treatment of, the diseases discussed herein.

A typical method for assessing the ability of an agent to modulate theaggregation and\or proteolytic processing of a precursor protein to aproduct in response to interaction with a template fragment, maycomprise:

-   (a) providing a stable cell or cell line as discussed above,-   (b) subjecting the cell to the stimulus such that the precursor    protein is expressed in the cell and whereby interaction of the    template fragment with the precursor protein causes a conformational    change in the protein such as to cause aggregation and proteolytic    processing of the precursor protein to a product fragment,-   (c) monitoring the production of the product fragment in the    presence of the agent,-   (d) optionally comparing the value obtained in step (c) with a    reference value.

The reference value may be based on historical observation, or may bebased on control experiments carried out in parallel e.g. in which oneinteger of the assay (template fragment, precursor protein, stimulus,agent) is modified or absent.

The various methods described above may comprise the further step ofcorrelating the result of step (d) with the modulatory activity of theagent(s).

Thus a method of identifying a modulator of aggregation of a proteinassociated with a disease in which the protein undergoes an inducedconformational interaction, may comprise performing a method forinducing aggregation as described above in the presence of one or moreagents suspected of being capable of modulating (e.g. inhibiting orreversing) the aggregation. The degree of aggregation (and optionallyproteolytic processing) may be observed in the presence or absence ofthe agent, and the relative values correlated with its activity as amodulator.

For example, a test substance may be added to a cellular system asdescribed above, and the cells incubated for a period of time sufficientto allow binding and to demonstrate inhibition of binding. The bound taucomplex can then be detected, e.g. using a suitably-labeled antibodysuch as MAb 7.51 in an immunoblot of total cell extract, or any othersuitable detection method.

Where a screening method is employed for this purpose, i.e. for theidentification of modulatory/inhibitory compounds, a non-competitive orcompetitive assay may be used. For instance, in a competitive assay ofthe type well known in the art, the effect of a known inhibitor ormodulator can be compared in the presence or absence of further testsubstances or agents, to determine the ability of the test substance tocompete with the known inhibitor/modulator for binding to the protein ofinterest.

Also provided are methods of producing modulators (e.g. inhibitors)which are as described above, but which further comprise the step ofproducing the modulator this identified.

Specificity of Inhibition

Screening methods according to this aspect of the present invention maybe used to screen for compounds which demonstrate the properties ofselective competitive inhibition of disease-related protein aggregation(e.g. tau-tau, tau-MAP2, or other protein, binding), withoutinterference with any ‘normal’ binding in which the precursor proteinparticipates (e.g. tau or MAP2 to tubulin, or by analogy, otherprecursor proteins with their binding partners insofar as these areknown).

Specifically in the case of tau, a method for determining any possibleinterference of the binding of tau, MAP2 or a derivative thereof totubulin by potential inhibitors/modulators, comprises contacting apreparation of depolymerised tubulin or taxol-stabilised microtubuleswith the agent, followed by detection of the tau-tubulin or MAP2-tubulinbinding. Tau-tubulin binding could also, for example, be demonstrated bya normal cytoskeletal distribution, as described in e.g. WO 96/30766.Methods for the preparation of tubulin proteins or fragments thereof,possibly in combination with binding partners, are known in the art andare described e.g. by Slobada et al. (1976, in: Cell Mobility (R.Goldman, T. Pollard and J. Rosenbaum, eds.), Cold Spring Laboratory,Cold Spring Harbor, N.Y., pp 1171-1212).

Analogous methods for other proteins having ‘disease’ and ‘normal’functions will occur to those skilled in the art in the light of thepresent disclosure.

Cell Viability

Where desired, methods of the present invention may further include thestep of testing the viability of the cells expressing the templateprotein and optionally precursor protein e.g. by use of a lactatedehydrogenase assay kit (Sigma).

In the case where tau-tau, tau-MAP2 or MAP2-MAP2 aggregation is beinginvestigated (see above, under ‘specificity’), this step may alsoprovide an indication of any interference by the test agent of thebinding of tau or MAP2 to tubulin, since inhibition or interference oftau-tubulin or MAP2-tubulin binding will correlate to some extent with adecreased ability of the cells to divide, and thus with decreased cellviability.

Cell viability may be used to derive an LD50 value for the agent.Preferred inhibitors will have a therapeutic index (LD50/B50—seediscussion of FIG. 9) of at least 2, 5, 10, or 20.

Choice of Test Agent

Compounds which are tested may be any which it is desired to assess forthe relevant activity.

The methods can serve either as primary screens, in order to identifynew inhibitors/modulators, or as secondary screens in order to studyknown inhibitors/modulators in further detail.

Agents may be natural or synthetic chemical compounds. Antibodies whichrecognise an Alzheimer's disease-like protein aggregate and/or whichmodulate Alzheimer's disease-like protein aggregation form one class ofputative inhibitory or modulatory compounds with respect to theaggregation process. More usually, relatively small chemical compounds,preferably which are capable of crossing the blood-brain barrier, willbe tested. Other qualities which it may be desirable to establish inconjunction with (before, simulataneously with, or after) use of thepresent invention, include: non-toxic to bone marrow, minimaldeleterious cardiovascular activity; minimal liver and renal toxicity;good oral absorption; non-metabolised to inactive form, and so on. Asthose skilled in the art are aware, these tests can be performed on acommercial basis by well established methods for compounds which it isdesired to test in this way.

For a typical test substance and putative modulator, where possible, thesolubility will first be determined e.g. from The Merck Index. Where thesubstance is soluble in aqueous solution, a concentrated stock solutionmay be prepared e.g. at 5-20 mM in PBS. Immediately prior to use thiscan be diluted with tissue culture medium to give a working stocksolution e.g. at 100 μM and introduced to cells to give a finalconcentration of between 0-10 μM for most compounds. Naturally, if it isdesired to test compounds at a concentration greater than 10 μM, theconcentration of the working stock solution may be increasedappropriately.

Where the substance is not soluble in aqueous solution, stock solutionsmay be made in an appropriate solvent (determined from The Merck Indexor experimentally) e.g. ethanol at 5-29 nM. This can again be dilutedwith tissue culture medium immediately prior to use to give a workingsolution e.g. at 100 μM concentration, and added to cells to yield afinal concentration of e.g. 0-10 μM for most test compounds. As above,if compounds are to be tested at a concentration greater than 10 μM theconcentration of the working solution will be increased as appropriate.

The skilled person will appreciate that the amount of test substance orcompound which is added in a screening assay according to this aspect ofthe invention, and indeed the manner in which it is introduced, can bedetermined by those skilled in the art, if necessary by use of a seriesof trials. Where the administered compound and the cell line haveconflicting optimal conditions (e.g. in terms of pH, or ionic strengthetc.) a variety of conditions should be tried to find an optimal,compromise, level. Initial concentrations may be selected to be a levelwhich could realistically be used in therapeutic context i.e. would benon-lethal to a patient (see comments on dosages below). In the light ofthe present disclosure, such an approach will not present any undueburden to one skilled in the art.

Screening Phenothiazines

The present invention extends, in further aspects, to compoundsidentified by a screening method as provided herein, and to compositionscomprising such inhibitors/modulators of induced conformationalpolymerisation of a protein.

As described in e.g. WO 96/30766, amongst the agents found to be able toinhibit pathological induced conformational polymerisation of proteinssuch as tau are certain diaminophenothiazines. Examples include such asthionine, methylene blue (MB), tolonium chloride, and dimethyl-methyleneblue (DMMB) which are of particular interest as potential therapeuticagents for use in the prevention of tau-tau aggregation in diseases suchas Alzheimer's Disease.

Interestingly, as described in more detail in the Examples, the presentinventors have used the methods described herein to demonstrate that themechanism of action of compounds such as MB on induced conformationalpolymerisation such as tau-tau aggregation is primarily steric innature. Additionally, it has been shown that the potent stericinhibitory effect, e.g. of the diaminophenothiazines on tau-tau binding,is dependent on the diffusion coefficient of the compound. The variousimplications of these observations in terms of screening and formulatingcompounds are discussed in more detail below.

This finding is particularly unexpected when considering the descriptionof the use of the such compounds in the prior art. Thus, for example,such compounds were previously known to be useful in the treatment ofmethaemoglobinaemia, where their action has been shown to be mediated bythe catalytic reduction of oxidised haemoglobin by transfer of electronsfrom the cell's intrinsic supply of reduced pyridine nucleotides (see,e.g. Hauschild, F. (1936) Arch. Exp. Pathol. Pharmacol. 182:118;“Pharmacological Basis of Therapeutics”, First Edition (1941), Goodmanand Gilman; Hrgovic, Z. (1990) Anästh. Intensivther. Notfallmed. 25:172; and Cudd, L. et al. (1996) Vet Human Toxicol. 38(5): 329) and inthe prophylaxis of manic depressive psychosis (Narsapur, S. L. (1983)Journal of Affective Disorders 5:155; Naylor, G. J. (1986) Biol.Psychiatry 21:915). Notwithstanding this, MB, thionine and toloniumchloride are actually intrinsically weak oxidising agents and, in theabsence of a supply of reduced pyridine nucleotides, they oxidiseproteins such as haemoglobin (Morse, E. (1988) Annals of Clin. Lab. Sci.18(1):13). This toxic effect can be used to inactivate viruses, and MBhas consequently been exploited therapeutically in a process forremoving HIV and hepatitis virus from blood products (Chapman, J.(1994), Transfusion Today 20:2; Wagner, S. J. (1995) Transfusion35(5):407). The mechanism of action of this effect is thought to involveintercalation of MB into DNA. The compound is boosted to a higher redoxstate by photoactivation and, when it drops back down to its groundstate, produces singlet oxygen which oxidises the DNA and inactivates it(Ben-Hur, E. et al. (1996) Transfusion Medicine Reviews, Vol. X, No. 1:15; Margolis-Nunno, H. et al. (1994), Transfusion 34(9): 802).Exploitation of the toxic effect of photoactivated diaminophenothiazineshas also been suggested for the treatment of cancer. Within cells,compounds which have been photoactivated to the oxidised form can damagemitochondria (Darzynkiewicz, Z. et al. (1988), Cancer Research 48: 1295)and/or microtubules (Stockert, J. et al. (1996) Cancer Chemother.Pharmacol. 39: 167).

Thus, on reviewing the prior art, it is apparent that two possiblemechanisms have been proposed to account for the action of compoundssuch as MB and thionine on entities such as DNA or proteins. The firstis the catalytic reduction of e.g. oxidised proteins by means oftransfer of electrons from reduced pyridine nucleotides in the cell. Thesecond proposed mechanism is the oxidation, and consequent inactivationof e.g. DNA by a photoactivated, oxidised form of compounds such as MB.In the light of these two mechanisms, it could therefore reasonably havebeen assumed that the inhibitory effect on tau-tau association ofcompounds such as MB was also attributable to a redox activity. That is,it might be assumed that such compounds inhibit induced conformationalpolymerisation such as tau-tau association by acting as weak oxidisingagents or as catalytic reducing agents.

Thus the work of the present inventors, in demonstrating that themechanism of action is primarily steric in nature, has unexpectedimplications for the choice, assessment, formulation and use of suchcompounds in the context of the diseases discussed herein.

In particular, certain compounds have been identified as feasibletherapeutics which would have been dismissed based on the result ofprior art assays. Specifically, Wischik et al. 1996 (loc cit) reportedon page 1217 that the concentration of MB required for inhibition washigher than could be achieved clinically. However the results hereinshow that the reduction of MB modifies its stacking ability in such away as to enhance its inhibitory potential to a level at which itbecomes clinically relevant for the treatment of e.g. tau aggregationassociated disease. This is discussed in more detail below in relationto the embodiments of the invention concerned with measurement ofdiffusion coefficients (which are also determined, in part, by thecompound's ability to ‘stack’).

FIG. 8 shows the structure of only some of the compounds which have beentested in the cell based assay. FIGS. 9-16 demonstrate the increasedpotency of certain compounds in the reduced form, plus some controlcompounds.

Thus in one aspect of the present invention there is disclosed use, inthe treatment of a disease disclosed herein, of a reduced (‘leuco’)phenothiazine of the formula:

wherein R₁, R₃, R₄, R₆, R₇ and R₉ are independently selected fromhydrogen, halogen, hydroxy, carboxy, substituted or unsubstituted alkyl,haloalkyl or alkoxy;

-   R₅ is selected from hydrogen, hydroxy, carboxy, substituted or    unsubstituted alkyl, haloalkyl or alkoxy; and each R₁₀ and R₁₁ are    independently selected from hydrogen, hydroxy, carboxy, substituted    or unsubstituted alkyl, haloalkyl or alkoxy;    or a pharmaceutically acceptable salt thereof.

Preferably, R₁, R₃, R₄, R₆, R₇ and R₉ are independently selected from-hydrogen, —CH₃, —C₂H₅ or —C₃H₇;

-   each R₁₀ and R₁₁ are independently selected from hydrogen, —CH₃,    —C₂H₅ or —C₃H₇; and-   R₅ is hydrogen, —CH₃, —C₂H₅ or —C₃H₇.

Preferably, the compound is a diaminophenothiazine which has 0, 2, 3 or4 methyl groups around the diaminophenothiazine nucleus. Preferably, thediaminophenothiazine is asymmetrically methylated (e.g., toloniumchloride, azure A, azure B and thionine).

Preferably the compound is selected from Methylene Blue, Toloniumchloride, Thionine, Azure A, Azure B or 1,9-Dimethylmethylene Blue.

Phenothiazines for use in the present invention may be manufactured bythe processes referred to in standard texts (e.g. Merck Manual,Houben-Weyl, Beilstein, E. III/IV 27, 1214 ff, J. Heterocycl. Chem. 21,613 (1984))

Instead of administering these compounds directly, they could beadministered in a precursor form, for conversion to the active form byan activating agent produced in, or targeted to, the cells to betreated. For instance, methylene blue may be administered in a precursorform, or it may itself serve as a precursor of the compound Azure A.

Stabilisation of Reduced Form

Some of these compounds of interest are known to circulate in the bodypredominantly in the reduced form. For example, for a discussion of thepharmacokinetics of MB, see e.g. DiSanto, A. et al. (1972) JournalPharm. Sci. 61(7):1086 and DiSanto, A. et al. (1972) Journal Pharm. Sci61(7):1090. Thirdly, only the reduced form of compounds such as MB isfound to cross the blood-brain barrier (Chapman, D. M. (1982) Tissue andCell 14(3):475; Müller, T. (1992) Acta Anat. 144:39; Müller, T. (1994)J. Anat. 184:419; Becker, H. et al. (1952) Zeitschrift fürNaturforschung 7:493; Müller, T. (1995) It. J. Anat. Embryol.100(3):179; Müller, T. (1998) Histol. Histopathol. 13:1019).

Such references as these illustrate that the reduced form of compoundssuch as MB represents a feasible and pharmaceutically-acceptableformulation for administration to subjects. MB has previously been usedclinically in an oral preparation. Further toxicological tests are,however, required before its clinical acceptability is achieved. Thehalf live of MB and related compounds (e.g. tolonium chloride) in bloodis approximately 100 minutes. It is evident that slow releaseformulations of compounds with such, relatively short, half lives cansubstantially improve compound availability and hence therapeuticefficacy.

FIG. 17 shows that compounds such as those discussed herein differgreatly in their extent of reduction in the conditions of the assay(approx. 500:1 DTT excess, at 120 minutes). As this figure shows,thionine is completely reduced under these conditions, tolonium chlorideis reduced at an intermediate level, and MB and DMMB are relativelylittle reduced. The amounts of commonly used reductant required toachieve, say, 90% reduction of the oxidized form in 10 minutes, prior toadministration\absorption may not be feasible (e.g. 2000:1 ratio of DTTto MB).

As FIG. 18 illustrates, the extent of reduction of MB underphysiological conditions can be greatly accelerated by allowingreduction over night and then lyophilising the reduced form. Thelyophilisate becomes reduced by 90% in 10 minutes, after solubilisationin conditions mimicking gastric acidity (5 mM HCl). Capsules containinga form of the diaminophenothiazine pre-reduced with ascorbic acid at amg ratio of 1.5-2 represent a suitable, if not optimal, formulation fortherapeutic use.

The same considerations apply to other compounds, such as thionine andtolonium chloride, which are more readily reduced than MB, but theextent of reduction of which can be accelerated in a manner such as thatdescribed above.

Thus in preferred forms the phenothiazine agents of the presentinvention are provided as pre-reduced compounds e.g. in lyophilisedpreparations, optionally in the presence of a stabilising agent.

An agent for stabilising the preferred form of the active compound (i.e.a form of the compound having a low diffusion coefficient, e.g. thefully-reduced form of the compound) may be a reducing agent orantioxidant. The agent may serve both to convert one form of theinhibitory compound (e.g. the oxidised form) to the preferred formthereof (e.g. the reduced form), and to stabilise that preferred (e.g.reduced) form. Alternatively, the inhibitory compound may be added tothe composition in its preferred (e.g. already-reduced) form, so thatthe agent merely serves to maintain the compound in this form.

Particularly suitable for use in converting to, and/or stabilising, thereduced form of the active agent (e.g. the diaminophenothiazine)comprised in the formulations of the present invention is theantioxidant ascorbate. Ascorbate has previously been used to minimiseoxidative damage of proteins (Parkkinen J. (1996), “Thrombosis andHaemostasis” 75(2): 292). A formulation as provided herein could thusadvantageously comprise a diaminophenothiazine, especially MB, toloniumchloride, DMMB or thionine, in combination with ascorbate, in suitableproportions, concentrations and dosages.

In other embodiments the reduced (leuco) form may be favoured by theaddition or selection of appropriate constituent groups.

Thus aspects of the invention further include a method of preparing amedicament for use in the treatment or prophylaxis of a disease asdescribed above, which method comprises the step of reducing thecompound (such that it is, say, at least 50, 60, 70, preferably 80, 90,95, or 99% reduced) and stabilizing it in a lyophilized composition inthe reduced form, prior to administration of an appropriate dose to apatient in need of the same.

Dosage of Therapeutics

Administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofthe disease being treated. Prescription of treatment, e.g. decisions ondosage etc., is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.

CNS penetration of MB following systemic administration has beendescribed by Müller (1992; Acta Anat. 144:39). Azure A and B are knownto occur as normal metabolic degradation products of MB (Disanto andWagner (1972a) J. Pharm. Sci. 61: 598; Disanto and Wagner (1972b) J.Pharm. Sci. 61: 1086). The pharmacokinetics and toxicity of toloniumchloride in sheep is discussed by Cudd et al (1996) Vet Human Toxic 38(5) 329-332.

For thionine, which is specifically exemplified herein, a daily dosageof between 1 and 1000 mg may be suitable, preferably divided into 1 to 8unit doses, which can, for example, be of the same amount. It will,however, be appreciated that these limits given above can be departedfrom when required, as may be appropriate with the compounds of theinvention other than thionine, which have higher or lower activity orbioavailability.

FIG. 19 shows the variation of tissue levels of MB vs IV dose.

The pharmacokinetics of methylene blue have been studied in humans, dogsand rats by DiSanto and Wagner, J Pharm Sci 1972, 61:1086-1090 and 1972,61:1090-1094. Further data on urinary excretion in humans is alsoavailable from Moody et al., Biol Psych 1989, 26: 847-858. Combiningdata on urinary excretion of MB in humans, it is possible to derive anoverall model for distribution of MB following single 100 mg dose in a70 kg subject, assuming instantaneous absorbtion (FIG. 19B). Urinaryexcretion accounts for 54-98% of the ingested dose. This variability ismost likely due to variability in absorbtion, although variability inmetabolism cannot be excluded. From urinary excretion data, it ispossible to calculate that whole body clearance is 56 mg/kg/hr.Therefore, the dosage required to achieve an effective target tissueconcentration of 4 μM is 1.73 mg/kg/day (0.58 mg/kg tds) if there werecomplete absorbtion. However, from Moody et al., it is clear that totalurinary excretion, and hence effective bioavailability, is itself afunction of dose. The oral dose required to deliver 1.73 mg/kg/day isapproximately 2× the dosage calculated on the basis of whole-bodyclearance. Therefore the actual required dosage is on the order of 3.2mg/kg/day. This is close to the minimum routine oral dosage usedclinically in humans, eg in the treatment of chronic urinary tractinfection (390 mg/day). The maintenance oral dosage in humans istherefore approximately 225 mg/day, or 75 mg tds. Peak tissue levels arereached at approximately 1 hr and the tissue half-life is about 12hours.

Methylene blue exists in the charged blue oxidised form, and theuncharged colourless reduced leukomethylene blue form. We have shownexperimentally in cells that the target tissue concentration in cellsrequired to prevent tau aggregation by 50% (ie the EC50) is 4 μM forreduced methylene blue, and that it is the leuko-form which ispreferentially active. It is shown by DiSanto and Wagner (1972) thatapproximately 78% of the methylene blue recovered in urine is in thereduced form, and from anatomical studies following iv administration,the only form which is bound to tissues is the colourless reduced form,which becomes oxidised to the blue colour on exposure to air afterpost-mortem dissection. The only form of methylene blue which crossesthe blood-brain barrier after iv administration is the reduced form(Muller, Acta Anat 1992, 144:39-44 and Becker and Quadbeck, 1952).Therefore, orally absorbed methylene blue is very rapidly reduced in thebody, and remains so until excretion, possibly undergoing furtherchemical modification which stabilises it in a reduced form.

It is highly likely that variability in oral absorbtion is determinedlargely by the efficiency of initial reduction in the GI tract. One wayto achieve more reliable absorbtion is therefore be to pre-reducemethylene blue with ascorbic acid. We have shown from in vitro studiesthat this conversion is rather slow, so that it takes 3 hours to achieve90% reduction of methylene blue in water in the presence of 2× mg ratioof ascorbic acid. Therefore, the dosage of methylene blue which is mostlikely to ensure reliable absorbtion will be 3.5 mg/kg/day of methyleneblue pre-reduced for at least 3 hours in the presence of 7 mg/kg/day ofascorbic acid.

It is also possible that MB may be active at lower concentrations inman, and that a range of clinically feasible doses would be therefore 20mg tds, 50 mg tds or 100 mg tds, combined with 2× mg ratio of ascorbicacid in such a manner as to achieve more than 90% reduction prior toingestion.

Formulation and Administration of Therapeutics

Suitable compounds, such as those with a formula as shown above or theirpharmaceutically-acceptable salts, may be incorporated into compositionsof this aspect of the present invention after further testing fortoxicity. The compositions may include, in addition to the aboveconstituents, pharmaceutically-acceptable excipients, carriers, buffers,stabilisers or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material may depend on the route of administration.

Where the composition is formulated into a pharmaceutical composition,the administration thereof can be effected parentally such as orally, inthe form of powders, tablets, coated tablets, dragees, hard and softgelatine capsules, solutions, emulsions or suspensions, nasally (e.g. inthe form of nasal sprays) or rectally (e.g. in the form ofsuppositories). However, the administration can also be effectedparentally such as intramuscularly, intravenously, cutaneously,subcutaneously, or intraperitoneally (e.g. in the form of injectionsolutions).

Where the pharmaceutical composition is in the form of a tablet, it mayinclude a solid carrier such as gelatine or an adjuvant. For themanufacture of tablets, coated tablets, dragees and hard gelatinecapsules, the active compounds and their pharmaceutically-acceptableacid addition salts can be processed with pharmaceutically inert,inorganic or organic excipients. Lactose, maize, starch or derivativesthereof, talc, stearic acid or its salts etc. can be used, for example,as such excipients for tablets, dragees and hard gelatine capsules.Suitable excipients for soft gelatine capsules are, for example,vegetable oils, waxes, fats, semi-solid and liquid polyols etc.

Where the composition is in the form of a liquid pharmaceuticalformulation, it will generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay also be included. Other suitable excipients for the manufacture ofsolutions and syrups are, for example, water, polyols, saccharose,invert sugar, glucose, trihalose, etc. Suitable excipients for injectionsolutions are, for example, water, alcohols, polyols, glycerol,vegetable oils, etc.

Suitable excipients for suppositories are, for example, natural orhardened oils, waxes, fats, semi-liquid or liquid polyols etc.

Moreover, the pharmaceutical preparations may contain preserving agents,solubilizers, viscosity-increasing substances, stabilising agents,wetting agents, emulsifying agents, sweetening agents, colouring agents,flavouring agents, salts for varying the osmotic pressure, buffers, orcoating agents.

For intravenous, cutaneous or subcutaneous injection, or intracatheterinfusion into the brain, the active ingredient will be in the form of aparenterally-acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers and/orother additives may be included, as required.

A composition according to the present invention may be administeredalone, or in combination with other treatments, either simultaneously orsequentially, dependent upon the condition or disease to be treated.

In accordance with the present invention, the formulations providedherein may be used for the prophylaxis or treatment of Alzheimer'sdisease, motor neuron disease, Lewy body disease, Pick's disease orProgressive Supranuclear Palsy, or any other condition or disease inwhich induced conformational polymerisation of a protein is implicated(see FIG. 5). In particular, as described in detail below, theformulation may be used for the blocking, modulation and inhibition ofpathological tau-tau association.

Examples of the techniques and protocols mentioned above can be found in“Remington's Pharmaceutical Sciences”, 16th edition, Osol, A. (ed.),1980.

In a further aspect, the present invention relates to the use of acomposition of the preceding aspect, in the diagnosis, prognosis ortreatment of a condition in which induced conformational polymerisationof a protein is implicated. The condition may be a disease such asAlzheimer's disease, or any other condition of the type describedherein.

Use of Diffusion Constant as a Screen

As stated above, by converting a compound into, and/or stabilising itsreduced form, the inhibitory potency of the compound can be optimised.

However, as described in more detail in the examples hereinafter,surprisingly, the redox potential of a compound does not directlydetermine its inhibitory activity with respect to induced conformationalpolymerisation of proteins, and that, therefore, neither the oxidationmodel nor a catalytic reduction model are relevant to an understandingof the activity of compounds as tau-tau aggregation inhibitors.

The inventors have found that there is a strong inverse correlationbetween the inhibitory potential of a compound towards tau-tau bindingand the square or third power of its diffusion coefficient.

The diffusion coefficient is determined by the amount of stacking ofdischarged molecules at a cathode. Experimentally, this can be evaluatedby measuring the current flow in a redox cell at the reductionpotential. The diffusion coefficient is inversely correlated with thedegree of aggregation of the discharged (i.e. reduced) species withinthe Helmholtz layer forming at the cathode. These aggregates form bypi-bonded stacking interactions across the phenol ring systems.

In one model, the lower the diffusion coefficient, the higher thetendency to stack, and the more potent the compound is in inhibitinginduced conformational polymerisation of proteins such as tau-taubinding, as reflected by a low K_(i).

The stacking of diaminophenothiazines may be less favoured when themolecule is in the oxidised form, since this form is charged, and so canbe envisaged to repel other, like molecules. This phenomenon may thusexplain the greater efficacy of the reduced form ofdiaminophenothiazines in the inhibition of tau aggregation (see e.g.FIG. 9).

Thus an assessment of the diffusion coefficient (dependent on‘stackability’, which is in turn dependent on shape and charge) can be auseful step in the development of effective modulators. One suchsterically-relevant parameter is diffusion coefficient which can bediminished by providing diaminophenothiazines in their reduced form.

Thus, the present inventors teach herein that the efficacy of a compoundin the blocking, modulation or inhibition of induced conformationalpolymerisation of a protein (hereinafter referred to as “inhibitorypotency” can be tested in an assay method which includes the step ofmeasuring the diffusion coefficient of the compound.

Hence, in its most general form, the present invention provides a methodof screening for an agent that blocks, modulates or inhibits inducedconformational polymerisation of a protein, which method includes thestep of measuring the diffusion coefficient of the agent. The use of thediffusion coefficient value, and in particular the square or third powerof its diffusion coefficient, in assessing the inhibitory potency of aphenothiazine (e.g. as described above) for the treatment of a diseaseas described herein forms a further aspect of the present invention.

The step of measuring the diffusion coefficient of the test agent may beincorporated at any stage of a larger screening programme foridentifying or optimising putative or established modulators.

The larger method will typically further include assay steps asdescribed herein, or in the prior art (e.g. WO 96/30766). Thus, in thelatter case for instance, when one wishes to screen for agents whichblock, modulate or inhibit tau-tau aggregation, the method may includethe steps of contacting:

-   (a) a tau protein or a derivative thereof containing the tau core    fragment, with;-   (b) a substance to be tested for its ability to block, modulate or    inhibit tau-tau aggregation; and-   (c) a labelled tau protein or a labelled derivative thereof which is    capable of binding to the tau protein of step (a) or a tau protein    or a derivative thereof which is distinct from the tau protein of    step (a) and also capable of binding to the tau protein of step (a).

The diffusion coefficient may be measured by any suitable means, forinstance according to the method of Murthy and Reddy (J Chem Soc.,Faraday Trans J 1984, 80. 2745-2750). This publication also includedsome determined values of diffusion coefficients for phenothiazine dyesand its content is specifically incorporated herein by reference.

Thus, the diffusion coefficient may suitably be measured by cyclicvoltammetry in an aqueous acidic medium, whereby the magnitude ofcurrent flow in a redox cell is tested at the reduction potential of thecompound.

The method may include the step of performing further tests on theagent, e.g. to ascertain its specificity as an inhibitor or modulator ofinduced conformational polymerisation of a particular protein (e.g.tau), or to determine its pharmaceutical acceptability or suitability asan agent for administration to an animal.

The surprising teaching as provided herein, that the efficacy of anagent in blocking, modulating or inhibiting induced conformationalpolymerisation of a protein is dependent, at least in part, on thediffusion coefficient of the agent, can be utilised in the optimisationof an agent's efficacy. The present inventors have established that anagent's inhibitory potency towards induced conformational polymerisationof a protein is inversely related to the square or third power of itsdiffusion coefficient. In other words, the inhibitory potency of anagent can be optimised by providing the agent in a form in which itsdiffusion coefficient is minimised.

Thus, in a further aspect, the present invention concerns a method ofoptimising the efficacy of an agent in blocking, modulating orinhibiting induced conformational polymerisation of a protein, whichmethod includes the step of minimising the diffusion coefficient of theagent.

In a further aspect, the present invention provides a pharmaceuticalcomposition for the prophylaxis or treatment of a condition in whichinduced conformational polymerisation of a protein occurs, thecomposition comprising a compound which is provided in, or convertedinto, a form in which its diffusion coefficient is minimised.

This, and further, aspects of the invention will be better understood byreference to the following figures and experimental data, given only byway of example.

FIGURES

FIG. 1 shows a schematic illustration of the structure of a pairedhelical filament (top) and the immunochemistry of neurofibrillarytangles during progression of Alzheimer's disease (bottom).

FIG. 2 shows a conceptual scheme wherein critical nucleating factorsprovide a ‘seed’ which initiates tau capture, which then becomesautocatalytic.

FIG. 3 shows a putative pathogenic model of Alzheimer's disease.

Tau aggregation is a proximal process prior to failure of axonaltransport and consequent neuronal death. The tau aggregation cascade canbe triggered either by a seeding/nucleation event arising from upstreamchanges or from primary mutations in the tau gene.

FIG. 4 shows how induction of full-length tau can lead to its conversioninto the 12 kD fragment, provided there is some preexisting 12 kD tau inthe cell.

FIG. 5A and FIG. 5B shows a table listing proteins which play a role indiseases of protein aggregation. Also listed are the diseasesthemselves, the aggregating domain and/or mutation believed to beinvolved, and the putative (maximum) fibril subunit size. One or moreliterature references for each protein is given.

FIG. 6 shows a schematic illustration of the binding sites of variousmonoclonal antibodies to different forms of N- and C-truncated tau.

FIGS. 7 a-b shows the nucleotide (SEQ ID NO: 1) and predicted amino acidsequences (SEQ ID NO: 2) of a human tau protein isoform. The sequencewas deduced from cDNA clone htau40.

FIG. 8 shows the structures of thionine, tolonium chloride,chlorpromazine and tacrine.

FIG. 9 gives cellular assay data for diaminophenothiazines, and astructurally related anthroquinone along with apparent KI values,determined as described herein. In the Figures and Examples herein, afurther parameter, B50, has been calculated to express activity in amanner directly related to the conditions of the cell-based assay, andtherefore providing an indication of the tissue concentration whichwould be required to achieve the corresponding activity in vivo. The B50value is the concentration of test compound used in the cell assay atwhich relative production of the 12 kD band from full-length tau wasreduced to 50% of that observed in the absence of the compound. There isa simple linear relationship between apparent KI value and B50 value asfollows:Cellular B50=0.0217×KI

In order to compare the relative usefulness of compounds astherapeutics, it may be desired to calculate an LD50 value. Whereinhibitory properties are similar, preferred compounds for clinical usemay be those which have the highest LD50 value. A therapeutic index(RxIndx) may be calculated for each of compounds tested in the cellassays as follows:RxIndx=LD50/B50

Toxicity of the compounds may be measured by cell numbers after 24 hrsexposure to the compound using a lactate dehydrogenase assay kitTOX-7(Sigma Biosciences) according to the manufacturer's instructionsafter lysis of remaining cells. Alternatively a kit from Promega UK(CytoTox 96) may be used, again according to the manufacturer'sinstructions.

FIG. 10 shows the results of using reduced thionine in the presentinvention, based on a data set of 7 experiments. The observed cell datafor production of the 12 kD band can be fitted closely (ie observed vspredicted correlation coefficient>0.9), to a standard functiondescribing inhibition of tau-tau binding in vitro. To obtain this fit,two assumptions need to be made, which are consistent with results fromother cell-based and in vitro studies:

-   1) the intracellular concentration of tau is approximately 500 nM;-   2) the tau-tau binding affinity is 22 nM.    using these assumptions, the function for cellular activity    predicted via standard inhibition model is:    Activity=[tau]/([tau]Kd*(1+[thionine]/KI))    can be solved by standard numerical methods to derive a value for    apparent KI. As indicated, the value for the reduced form of    thionine is 100 nM. which is essentially the same as that observed    for tau-tau binding in vitro at a tau concentration of 500 nM, where    the Kd value for tau-tau binding is known to be 22 nM. Therefore,    the activity of thionine, where the read-out is production of the 12    kD truncation product from full-length tau, can be explained    quantitatively on the basis of extent of inhibition of the tau-tau    binding occurring through the repeat domain within the cell. This    confirms that the extent of tau-tau binding determines production of    the proteolytically stable core tau unit of the PHF within the cell.

All subsequent cellular analyses of activities of other compounds arereported in the same standardised format, with the same assumptionsregarding intracellular tau concentration (500 nM) and tau-tau bindingaffinity (22 nM) through the repeat domain.

FIG. 11 shows the results for conditions in which the reducing agentshave been omitted (i.e. oxidised thionine cf. FIG. 10).

Again cellular activity is predicted via standard inhibition model:Activity=[tau]/([tau]Kd*(1+[Ox.Thio.]/KI))

In this case, thionine now has an apparent KI value of 1200 nM. Thisconfirms that the diaminophenothiazines require to be in the reducedform for activity. A similar conclusion was derived from analysis of invitro binding data (results not shown).

FIG. 12 shows that by using reducing or partially reducing conditionsmethylene blue appears much more active in the cell-based assay thanpredicted from in vitro studies in which the time course of the assay(1-2 hours) had not been sufficient to achieve reduction.

Cellular activity is again predicted via standard inhibition model:Activity=[tau]/([tau]Kd*(1+[MB]/KI))

In the cell assay, the apparent KI value for methylene blue is 123 nM,which is within the same range as thionine and tolonium chloride. Asindicated in FIG. 9, the corresponding brain tissue concentration (i.e.B50 value) required to inhibit tau aggregation would be 2-3 μM.

FIG. 13 shows corresponding cell-based activity data for reducedtolonium chloride, indicating again that the predicted KI value derivedfrom in vitro studies can be used to describe production of the 12 kDfragment from full-length tau in cells.

Cellular activity is predicted via standard inhibition model:Activity=[tau]/([tau]Kd*(1+[TC]/KI))

This provides further confirmation of the validity of the mathematicalanalysis procedure used.

FIG. 14 shows that DH12 (anthroquinone) which is structurally related tothe diaminophenothiazines is inactive in the conditions of the assay.

FIGS. 15 & 16 show similar analyses to those given above in FIGS. 9-14,but for chlorpromazine and tacrine respectively. Using the sameassumptions (tau concentration 415 nM, and tau-tau binding Kd 22 nM),and cellular activity predicted via standard inhibition model:Activity=[tau]/([tau]Kd*(1+[cpz]/KI))the apparent KI values for chlorpromazine and tacrine (2117 nM and 802nM respectively) are greater than anticipated from the in vitro studies.

FIG. 17 shows the extent of reduction of various compounds in thepresence of DTT.

FIG. 18 shows the percentage reduction of MB plotted against the ratioof MB:Vitamin C.

FIG. 19A shows that by assuming a target tissue concentration of 4 μM(i.e. 1.5 μg/g) it is possible to determine from the data of DiSanto andWagner (1972) that tissue concentrations of this order would be achievedat an IV dosage of 0.11 mg/kg.

FIG. 19B shows a model for the distribution of MB following a single 100mg dose in a 70 kg subject, assuming instantaneous absorbtion.

FIG. 20 summarises the results for the transient expression of taufragments in 3T3 and COS-7 cells based upon data from both microscopicaland biochemical experiments.

Expression of recombinant tau fragments in eukaryotic cells wasperformed as follows. Eight tau constructs, transiently expressed in 3T3cells and COS-7 cells were examined by immunocytochemistry andimmunoblots. The extent of expression in each cell type was givensemi-quantitatively on the basis of both sets of results: −, nodetectable expression; ±, very weak immunoreactivity; + to ++++,increasing levels of positive immunoreactivity. In all cases, mAb 7.51was used with each construct to obtain the results. In addition thespecificity was confirmed for each construct by using a panel ofantibodies against different domains of tau protein (mAbs 499, T14,Taul, 342, 7.51, 423 and T46). Kozak sequences were absent in the firstsix contructs, but were present in the cDNA constructs 7 and 8.

FIG. 21 illustrates the inducible expression of full-length human tau in3T6 fibroblasts in two cell lines. T40.22 shows low level backgroundleakage of full length tau in the uninduced state (“U”), and high levelsof expression after addition of IPTG (i.e. induced, “I”). T40.37 showsthe same, but lower levels of expression without induction.

FIG. 22 shows a result of a triple vector system. A vector permittingvery low level constitutive expression of the 12 kD fragment wasintroduced into cells lines in which inducible expression of full lengthtau had already been achieved (in fact cell line T40.22 shown in FIG. 21above). Low levels of IPTG are introduced to induce expression offull-length tau. At 0 μM IPTG, there is very low level expression of the12 kD band, and low “background leakage” expression of full-length tau.As progressively more full-length tau is induced by introducing higherlevels of IPTG, more of the full-length tau is converted to the 12 kDspecies.

FIG. 23 shows the inhibitory effects of reduced thionine. In each set oflanes, there is inducible production of the 12 kD band in the presenceof increasing concentrations of IPTG inducing higher levels of T40. Asthe thionine concentration is increased, the production of the 12 kDband from T40 is suppressed.

FIG. 24 shows quantitatively the results of FIG. 23. In the absence ofthionine, induction of T40 at increasing concentrations of IPTG leads toa corresponding increased production of the 12 kD fragment. In thepresence of 2 μM thionine, there is still induction of T40, but it isnot converted into the 12 kD fragment.

FIG. 25 shows comparative in vitro KI values for various compounds, innM. The KI values relate to the particular assay conditions used (500:1DTT:compound, 120 minutes—see FIG. 17).

FIGS. 26 and 27 show the inhibitory effect on tau-tau binding ofphenothiazines having 0, 2, 3 or 0, 4, 6 methyl groups, respectively.

FIG. 28 shows the derivation of two parameters useful for measuring theinhibition of tau-tau association by test compounds. STB is thestandardised binding relative to that seen in the absence of compound,taken as the mean observed at 1 and 10 μg/ml. As described in WO96/30766, an STB value of 1.0 represents binding equivalent to thatobserved in the absence of compound, whereas a value of 0.2 indicatesthat the binding was reduced to a mean of 20% at test compoundconcentrations of 1 and 10 μg/ml. LB50 is log 10 molar ratio ofcompound:tau producing 50% tau-tau binding compared with that seen inthe absence of compound (B50).

FIG. 29 shows the relationship between STB and LB50 parameters. STB canbe shown to be a linear function of the LB50.

STB is a logarithmic function of the molar ratio of compound:tau atwhich tau-tau binding is reduced by 50%.

LB50 is the log of the molar ratio of compound with respect to tau atwhich tau-tau binding is 50% of that observed in the absence of compoundLB50=0.05+(2.65×STB) r=0.95

The determination of in vitro B50 requires that there be some degree ofinhibition of tau-tau binding, and a 50% value is obtained byextrapolation. Determination of STB requires no such extrapolationprocedure.

FIG. 30 shows compounds for which both STB and B50 values have beendetermined. Assuming that the total tau concentration in cells isapproximately 500 nM (i.e. the concentration of tau used in the assay),the B50 values provide an approximation in the in vitro assay to theconcentration (i.e. [500×B50] nM) at which the activity might beexpected in cell systems.

FIG. 31 shows the formal relationship between the in vitro LB50 valueand the log KI value for the diaminophenothiazine series.

FIG. 32 shows the relationship between the number of methyl groups in adiaminophenothiazine (NMETH) and the redox potential (E) and diffusioncoefficient (DIF). Italicised figures indicate correlation coefficients(R) and p values after exclusion of MB.

FIG. 33 shows the relationship between the percentage of compound thatis reduced, as determined experimentally, and the known reductionpotential of the compound. The reduction potential predicts the observedextent of reduction of the diaminophenothiazines.

FIG. 34A shows that there is no clear relationship between inhibitorypotency and the extent of reduction of compounds.

FIG. 34B shows that inhibitory potency is not determined simply byreduction potential.

FIG. 35 shows that the inhibitory potency can be related directly to thediffusion coefficient (which is a measure of the tendency of the reducedform to stack and aggregate).

FIGS. 36 and 37 show the predicted relationships between estimated LB50(“ESTLB50”) and STB (“ESTSTB”) values, respectively, and reductionpotential and diffusion coefficient, in which the diffusion coefficientis given the greater weighting.

FIG. 38 shows the crystalline structure of Methylene Blue.

FIG. 39 shows tau-tau binding in the presence of 1 mM DTT, as measuredin the solid phase assay of WO 96/30766. Two different antibodies wereused to detect tau-tau binding, namely mAb 342 (top) and 499 (bottom).The vertical axis represents tau-tau binding, the horizontal axis showsthe concentration of full-length tau in the aqueous phase, and the keyshows varying concentrations of solid-phase tau. As can be seen, tau-taubinding still occurs in the presence of DTT.

FIG. 40 shows various species of tau fragments and doublets which arepresent without induction (“U”) and following induction (“I”) in a cellline of the present invention. These include species with mobilitiesequivalent to 12/14 kD, ˜25/27 kD, ˜30/32 kD, ˜36/38 kD and ˜42/44 kD(see Example 3).

FIG. 41( a) shows how the 12 kD fragment arises via template-inducedproteolytic processing of full-length tau molecules at the approximatepositions shown by the arrow-heads.

FIG. 41( b) shows how the 25/27 kD species arises via template-inducedproteolytic processing of full-length tau molecules at the approximatepositions shown by the arrow-heads.

FIG. 42 shows a plot of the apparent gel mobilities of the species ofFIGS. 40-41 and their lengths in amino-acid residues.

FIG. 43 shows the fragments of FIGS. 40-42 are at intervals of either˜34 residues or ˜17 residues which is the equivalent of a single taurepeat, or half of it. All of the fragments may be generated from abasic heptameric aggregate as a simple set of proteolytic cleavagesoccurring at the positions indicated by the arrowheads.

FIG. 44 shows these same fragments in descending order of length andincreasing gel mobility.

FIG. 45 shows that DMMB is surprisingly potent in the cell model. Itsinhibitory activity could be seen both in the absence of IPTG inductionand following induction (see Example 4).

FIG. 46 shows the activity of DMMB on base-line expression of the 12/14kD species, using the same set of assumptions regarding intracellulartau concentration and in vitro tau-tau binding affinity used in FIGS.10-16.

Cellular activity is predicted via standard inhibition model:activity=[tau]/([tau]Kd*(1+[DMMB]/Ki))

DMMB has an apparent KI within the cell of 4.4 nM, and the cellular B50value is ˜100 nM.

EXAMPLES

General Materials and Methods

Production of 3T6H Cell Lines

3T6 cells were ECACC No: 86120801 Mouse Swiss Albino Embryo Fibroblasts.

For the inducible system, the experiments employed Lac Switch™ fromStratagene using the p3'SS vector to express the Lac repressor proteinand pOPRSVICAT to express the full-length tau under the control of theLac repressor. Expression is induced by the addition of IPTG.

Initially 3T6 cells were transfected, by electroporation, with the p3'SSplasmid and colonies selected by hygromycin resistance. 5 clones thatwere expressing varying levels of the Lac repressor protein (determinedby immunocytochemistry) were picked, and also the non-cloned cells wereretained for comparison.

Production of pOPRSVT40 Vector

The T40 insert for cloning into the pOPRSVICAT vector was prepared byPCR with Vent polymerase (NEB) using primers (shown below) that includeda Not I site and a start or stop codon as appropriate. The PCR productand pOPRSVICAT vector were cut with Not I and purified. The vector wasdephosphorylated to prevent re-ligation, and the insert ligated into thevector using standard protocols.

The resulting ligation mix was transfected into competent E. coli cellsand the cells plated out on amp plates. Colonies were picked and griddedout on a new amp plate. Colony lifts were taken to Hybond-N 0.45 μmnylon membrane (Amersham) and possible positives selected by colonyhybridisation using dGA labelled with (α-³²P) dCTP (Amersham) (using anoligolabelling kit (Pharmacia Biotech) and purified on a Nap-10 column(Pharmacia Biotech)). Hybridisation was carried out a 65° C. overnightin Church buffer followed by 2×20 mins washes in Church wash. Positivecolonies, labeled with radioactive probe, were detected by exposing theblots to x-ray film overnight at −70° C.

Positive colonies were selected and grown, then checked by PCR andrestriction digest to confirm the presence of the insert. The use of asingle restriction site for the cloning means that T40 can insert intothe vector in either orientation. The orientation of the inserts wasdetermined so as to select colonies with the vector containing T40 inthe correct orientation for expression.

Primers Used

5′-3′ T40-Not I                                start 5′-gtc gac tct agaggc ggc cgc ATG GCT GAG CCC CGG CAG GAG-3′                        Not I3′-5′ T40- Not I                                stop 5′-act ctt aag ggtcgc ggc cgc TCA CAA CAA ACC CTG CTT GGC CAG -3′                      Not I

Sequence complementary to T40 sequence is shown in capitals, the startand stop codons are marked. The Not I site to be added is shownunderlined. The remaining sequence shown in lower case is a 13 base pairoverhang to allow the Not I enzyme to cut efficiently. This wascomplementary to sequence in the hTau40 plasmid vector to allowefficient binding of the primers.

Determination of Insert Orientation

Orientation was determined using a restriction enzyme that cuts theinsert once and the vector at most a few times, and that gives adiffering restriction digest pattern for each orientation. Hind III fitsthese criteria for pOPRSVT40. If the insert is absent two restrictionbands are produced. If the insert is present three bands are producedand the size of the bands depends on the orientation of the insert asshown below.

Forward (correct) Orientation 5385 bp 1030 bp 381 bp Reverse Orientation6101 bp  381 bp 314 bpProduction of Cells Expressing T40 Under the Control of an InduciblePromoter

The pOPRSVT40 plasmid was produced and purified by CsCl gradientcentrifugation. This was transfected (by electroporation) into 3T6Hcells (expressing the Lac repressor protein) produced as describedabove. Positive cells were selected for by resistance to G418 (at 500μg/ml). Resistant colonies were picked and grown on. The level ofexpression of full-length T40 with and without the addition of IPTG wasdetermined with anti-tau antibodies by both immunocytochemistry andWestern blot.

Production of pZeo295-391

The plasmid pZeo295-391 was designed to express protein corresponding tothe truncated fragment of tau (residues 295-391; see below). Aconstitutive system (pcDNA3.1 from InVitrogen, Netherlands) was used—theplasmid imparts resistance to the antibiotic zeocin. The cDNA for thisregion was amplified by polymerase chain reaction (PCR), using specificoligonucleotide primers (sense and antisense; see below). The senseprimer contained an EcoRI site and the antisense, a BamHI site. Thefragments were subcloned into pcDNA3.1 (−)zeo (Invitrogen, Netherlands)that had been digested with EcoRI and BamHI. The inserted DNA isdownstream from a cytomegalovirus promoter sequence and upstream of apolyadenylation signal. The plasmid contains the DNA sequence for theexpression of ampicillin and zeocin resistance for selection in bacteriaand eukaryotic cells, respectively. The authenticity of the inserted DNAwas confirmed by full-length sequencing of both strands.

Nucleotide and amino acid sequence for truncated tau fragment 295–391gataatatcaaacacgtcccgggaggcggcagtgtgcaaatagtctacaaaccagttgacctgagcaaggtgacctccaagtgtggctcattaggcaacatccatcataaaccaggaggtggccaggtggaagtaaaatctgagaagcttgacttcaaggacagagtccagtcgaagattgggtccctggacaatatcacccacgtccctggcggaggaaataaaaagattgaaacccacaagctgaccttccgcgagaacgccaaagccaagacagaccacggggcggagDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAE 295 sense primer                      metasp²⁹⁵ 5′ - CGG AAT TCC ACC ATG GAT AAT ATC AAA CAC GTC CCG - 3′        EcoRI 391 anti-sense primer                   stop glu³⁹¹ 5′ - CGCG GGA TCC TCA CTC CGC CCC GTG GTC TGT CTT GGC - 3′             BamHI

The start and stop codons are in bold and the EcoRI and BamHIrestriction sites to be added are underlined.

Tissue Culture of Cells for Assay

The medium used was DMEM (with Glutamax I, pyruvate, 4.5 g/l glucose)from Life Technologies, Scotland. This was supplemented with 10% FCS(Helena BioSciences), 50 U/ml penicillin, 50 μg/ml streptomycin, plsufurther antibiotic as appropriate for the selection and maintenance ofthe relevant plasmid. Antibiotic concentrations were 200 μg/mlhygromycin (p3'SS selection and maintenance), 500 μg/ml G418(pOPRSVT40selection and maintenance), 400 or 200 μg/ml zeocin (pZeo295-391selection or maintenance).

Cells are grown at 37° C., in a humidified atmosphere of 5% CO₂. Cellsare maintained in 10 cm dishes, and split when they approach confluency.Medium is removed, cells washed with PBS and cells released bytrypsinisation with 1 ml of trypsin/EDTA solution/10 cm dish. Cells areresuspended in fresh medium at 1:10 dilution, or optionally in a rangeof dilutions from 1:5 to 1:20 (approximately 5000 to 20000 cells/cm²).

For the testing of drugs, cells are plated in 6 well or 24 well platesat an initial density that will allow them to grow to 50-80% confluencywithin 24 hours. Drugs are added to the well at various concentrations,expression of full-length tau is induced by the addition of IPTG at 0-50μM. Cells are grown for a further 24 hours and then collected foranalysis by SDS PAGE/Western blotting

Preparation of tau Protein

Recombinant tau (clone htau40) and perchloric acid-soluble tau extractedfrom rat and human brain were prepared as described previously (Goedert,M. & Jakes, R. (1990) EMBO J. 9:4225; Goedert, M. et al (1993) Proc.Natl. Acad. Sci. USA 90:5066).

Gel Electrophoresis and Blotting

Cells grown as outlined above are washed once with PBS then lysed in 50μl (24 well plates) or 100 μl (6 well plates) laemli buffer. Samples arestored at −20° C., boiled for 4 mins prior to running on 15% acrylamidegels using the BioRad miniProtean III mini gel system. Protein istransferred to PVDF membrane by Western blotting using the CAPs buffersystem. The membranes are incubated in block buffer (5% non-fat milkpowder (Marvel), 0.1% Tween 20 in PBS) for 1 hr to overnight. Tauprotein is detected by incubating the membranes with mAb 7.51 diluted1:5 with block buffer for 1-3 hrs or overnight, washing well withPBS/0.1% Tween20, incubating with anti-mouse HRP 1:5000 dilution inblock buffer for 1 hr, and washing well with PBS/0.1% Tween20. Boundantibody is detected by ECL reaction detected on ECL hyperfilm(Amersham).

Blots are scanned into a computer on a Hewlett Packard Scanjet 6100Cflatbed scanner at 600 dpi and saved as tiff files. Densitometry of theT40 and dGAE bands is performed with the Scananalysis program on anApple Power Mac G3.

Drug Preparation

Thionine, methylene blue, DMMB, and tolonium chloride are all preparedas a 1 mM stock in ddH2O. Prior to use a 100 μM dilute stock is preparedin HBSS which is added directly to the medium on cells.

For oxidised drug this is prepared simply by diluting the 1 mM stock inHBSS and filter sterilising.

For reduced drug the 1 mM is treated with ascorbic acid and DTT to yield0.5 mM drug, 50 mM ascorbic acid 50 mM DTT, this is allowed to stand for15 mins (turns blue to colourless) before making the dilute stock. Thisis diluted in HBSS to yield 100 μM drug, 10 mM ascorbic acid, 10 mM DTTand filter sterilised. Cells are treated with the drug at variousconcentrations, but for the reduced drug the ascorbic acid and DTTconcentrations are maintained at 400 μM throughout by using appropriatequantities of 100 μM reduced stock, 100 μM oxidised stock and 10 mMascorbic acid/DTT stock.

SDS Gel Electrophoresis and Immunoblotting

Standard electrophoresis and immunoblotting procedures were used asdescribed previously (Wischik, C. M. et al. (1988) Proc. Natl. Acad.Sci. USA 85:4506; Novak, M., et al. (1993) EMBO J. 12:365; Jakes, R. etal. (1991) EMBO J. 10:2725). Immunoblots were developed with the ABC kit(Vector Laboratories). The monoclonal antibodies (mAbs) 7.51, 21.D10,499 and 342 were used as undiluted hybridoma culture supernatant fluids.mAb AT8 (Innogenetics, Belgium) was used at 1/1000 dilution. Anti-taumAbs 7.51 (which recognises an epitope in the last repeat; see Novak, M.et al. (1991) Proc. Natl. Acad. Sci. USA 88: 5837), 423 (whichrecognises tau C-terminally truncated at residue Glu-391; see Wischik,C. M. et al. (1988) Proc. Natl. Acad. Sci. USA 85:4506; Novak, M. et al.(1993) EMBO J. 12:365), 499 (which recognises a human-specific tausegment between residues Gly-14 and Gln-26; see Wischik, C. M. et al.(1996) Proc. Natl. Acad. Sci. USA 93:11213), and 342 (which recognises asegment between residues Ser-208 and Pro-251). mAb 21.D10 was raisedagainst the A68-tau brain extract (Lee, V. M.-Y. et al. (1991) Science251: 675).

Tau Binding Assay

This was carried out basically as described in Wischik, C. M., et al.(1996) Proc. Natl. Acad. Sci. USA 93:11213. Solid phase tau (0-20 μg/ml)was coated on 96-well poly(vinyl chloride) microtitre plates in 50 mMcarbonate buffer at 37° C. for 1 h. The plate was washed twice with0.05% Tween 20, then blocked with 2% Marvel in PBST for 1 h at 37° C.After washing again, the plate was incubated for 1 h at 37° C. withaqueous phase tau (0-300 μg/ml in PBST containing 1% gelatin). In thepresent application, 1 mM DTT was also added.

The plate was washed twice and incubated for 1 h at 37° C. with mAb 499or 342, diluted with an equal volume of 2% Marvel in PBST. Afterwashing, horseradish peroxidase-conjugated goat-anti-mouse antibody (1/1000 in PBST) was incubated for 1 h at 37° C. The plate was washed andincubated with substrate solution containing tetramethylbenzidine andH₂O₂ and the rate of change of absorbance measured using a V_(max) platereader (Molecular Diagnostics, California) as described previously(Harrington, C. R. et al. (1990) J. Immunol. Meth. 134:261). Eachexperiment was performed in triplicate and included controls in whichboth solid phase and aqueous phase tau were absent, and also with eitherone of the two absent.

Data Analysis

This was performed as described in Wischik et al. (supra) and curveswere fitted according to the Langmuir equation with the Kaleidagraph(Synergy, Philadelphia) or Systat (SPSS Inc., Chicago) programs usingquasi-Newton approximation. Curve-fitting correlation coefficients aregiven in the Figures.

Example 1 Constitutive Expression of Full-Length, Truncated and Mutatedtau

Expression of tau in eukaryotic cell lines was sought to generate acellular model of tau aggregation under physiological conditions whichdid not suffer from the limitations of lipofectin-based approaches. Thisinvolves the expression of full-length tau and truncated tau fragmentsfor both normal tau and tau carrying pathogenic mutations.

Full Length tau

When normal full-length tau (T40) was transfected into cells (3T3 andNIE-115) it was expressed and involved in the assembly of themicrotubule network within the cells.

Truncated tau

Initially the cDNA for truncated tau fragment from the core of the PHF,corresponding to fragment 297-391, was transfected into non-neuronal 3T3fibroblasts: this truncated tau was selected since it is: (i) present inthe PHF-core; (ii) detected as deposits in AD brain tissue during theearly stages of the disease; (iii) capable of supporting the catalyticcapture and propagation of tau capture in vitro. Subsequently, a seriesof transfections was performed in which the extent of truncation ateither N- or C-termini was varied, based partly on the immunochemicalproperties of the tau molecule. Six constructs were created withtruncation at the N-terminus (186-441; 297-441) at the C- and N-termini(186-391; 297-391) and at the C-terminus (1-391). The pattern ofimmunoreactivity for the six constructs with a limited panel ofantibodies was capable of discriminating all of the tau fragmentsgenerated in this way.

The constructs were expressed in eukaryotic cells both transiently(using pSG5 as the vector) and stably (using pIF2 and pZeo as vectors).Stable transfectants are selected on the basis of resistance to theantibiotics geneticin and zeocin for pIF2 and pZeo, respectively.Epitope analysis was performed on bacterially expressed proteins usingpRK172 as the vector. FIG. 20 summarises the results for variousfragments in 3T3 and COS-7 cells. Further results showed that theexpression of two forms of tau in the same cell can affect the patternof immunoreactivity. For example, stable expression of 1-391 and 295-391results in the appearance of abnormal bundles within the cells. However,maintaining such cells in a stable and reproducible state provedelusive.

Mutated tau

Mutagenesis of full-length tau was used to generate known clinicalmutations. These were subcloned into pIF2 and stable transfectantsgenerated in 3T3 and NIE cells for a number of mutations including thosewhich affect microtubule assembly properties of tau (G272V, V337M, P301S, R406W) and S305N, which affects the alternative splicing of the taugene in vivo. In general, cells expressing full-length tau carryingmutations exhibited labelling of the microtubular network and wasindistinguishable from cells transfected with wild-type tau. Cell linesexpressing certain truncated tau fragments including mutations provedunstable.

CONCLUSION

In summary, the constitutive expression of truncated tau withineukaryotic cells proved difficult to achieve. Although transienttransfection systems permitted the optimisation of expression of tau bymanipulating the Kozak consensus surrounding the initiation codon for297-tau, the expression of e.g. 297-391 was still modest, suggestingsome inherent toxic properties of the fragment. Stable transfectionsreiterated this conclusion. This latter system demonstrated thattruncation at either N- or C-termini resulted in a slightly greaterpropensity for the tau to assemble in amorphous deposits rather than ina microtubular network. Stable expression of combinations of taufragments also generated aggregates within the cytoplasm of cells, butthis system was not readily reproducible.

Example 2 Inducible Expression of Truncated tau

In a further attempt to create a stable, reproducible system, withoutthe toxicity associated with constitutive expression, inducibleexpression of the core-tau fragment of the PHF (i.e. 297-391—which is 12kD) was attempted.

Several inducible systems for expression of proteins in eukaryotic cellswere tried, although the preferred system was the “lac switch” system.In this system, two vectors are incorporated into cells, typically 3T3or 3T6 fibroblasts which do not express any endogenous tau protein. Thefirst, the p3'SS vector codes for constitutive expression of the lac Igene, and expressors are selected on the basis of hygromycin resistance.The second, pOPRSVICAT incorporates the DNA coding for the tau proteinfragment under the control of a strong RSV promoter which containsoperator sequences from the Lac operon. Cells which incorporate thisvector are selected on the basis of neomycin resistance. Cells whichhave incorporated both vectors have the property that constitutiveexpression of lac I prevents expression of the incorporated protein(i.e. tau) controlled by the Lac operon. The addition of the sugar IPTGcompetes for the binding of lac I to the Lac operon, and so permitsexpression of tau protein.

Inducible expression of the 12 kD fragment was carried out in two celllines. These did not show appreciable levels of tau protein expressionuntil after 3 days treatment with IPTG at which stage high levels of 12kD suddenly appeared, forming intracellular aggregates which promptlykilled the cell. The process of aggregation was, as expected, non-linearprogressing from low level expression to sudden accumulation of toxicaggregates without any clear gradation, making the aggregation andtoxicity impossible to control. This non-linear progression prevented aproper control of the system.

EXAMPLE 3 Expression of tau in Stable Cell Lines According to Invention

In view of the results above, a further system was used as follows.Tissue culture cell line DH 19.4.1.4 and clones thereof were based on3T6 cells (ECACC No: 86120801 Mouse Swiss Albino Embryo Fibroblasts)expressing full-length, four repeat human tau under the control of aninducible promoter and truncated human tau (295-391) under the controlof a constitutive promoter.

Cells expressing T40 under the control of an inducible promoter,T40.22.10, were transfected (by lipofection) with the pZeo295-391plasmid. Positive cells were selected for by resistance to zeocin at 400μg/ml. Expression of truncated tau on a background of inducibleexpression of full-length tau was confirmed by Western blot analysiswith Mab 7.51.

FIG. 21 illustrates the inducible expression of full-length human tauonly in 3T6 fibroblasts in two cell lines. T40.22 shows low levelbackground leakage of full length tau in the uninduced state (“U”), andhigh levels of expression after addition of IPTG (i.e. induced, “I”).T40.37 shows the same, but lower levels of expression without induction.FIG. 22 shows the results of a triple vector system. A vector permittingvery low level constitutive expression of the 12 kD fragment wasintroduced into cell lines in which inducible expression of full lengthtau had already been achieved (T40.22 shown in FIG. 21). FIG. 22 showswhat happens when low levels of IPTG are introduced to induce expressionof full-length tau. At 0 μM IPTG, there is very low level expression ofthe 12 kD band, and low “background leakage” expression of full-lengthtau. As progressively more full-length tau is induced by introducinghigher levels of IPTG, more of the full-length tau is converted to the12 kD species, and more of the intermediate higher molecular weightfragments (described in more detail in FIGS. 43 and 44) are produced.

Examination of the original T40-inducible cell line (T40.22.10) whichdid not contain the vector for constitutive expression of the 12 kDfragment shows that the 12 kD species is not produced as a truncationby-product of full-length tau induction. Enhanced expression of the 12kD band following induction of T40 was seen only in cells with low levelprior expression of the 12 kD fragment (DH19.4.1.4.6). That is,pre-existing 12 kD provides a template for production of more 12 kDfollowing the induction of full-length tau. An additional doublet mayalso appear with apparent gel mobility of −25/27 kD when the cells arein the uninduced state (e.g. in the cell line designated DH19.4.1.4A.B2). Following induction with IPTG, a further series ofdoublets may appear, with gel mobilities ˜30/32 kD, ˜36/38 kD and ˜42/44kD.

These species are shown in FIG. 40 both without induction (“U”) andfollowing induction (“I”). Also shown are the patterns ofimmunoreactivity of these fragments seen with mAb 342 and a C-terminalpolyclonal antibody T46 which recognises epitopes located betweenresidues Ser422 and Leu441.

The derivation of the fragments seen in the uninduced state (i.e. 12/14kD and 25/27 kD) may be explained by reference to FIG. 41.

FIG. 41( a) shows how the 12 kD fragment arises via template-inducedproteolytic processing of full-length tau molecules at the approximatepositions shown by the arrow-heads.

In the case of the 25/27 kD species, these fragments cannot representdimers of the the 12/14 kD species, as these fragments areimmunoreactive with T46. Therefore, a further proteolytic product of thefull-length aggregating tau protein must arise via cleavages occurringat the approximate positions shown by the arrowheads in FIG. 41( b).

Following induction (FIG. 40, I), the further series of doublets isseen. The derivation of these further fragments can be better understoodwith reference to FIGS. 42-44.

FIG. 42 shows a plot of the apparent gel mobilities of these fragmentsand their lengths in amino-acid residues, indicating that the apparentgel mobilities can be understood in terms of a characteristic set offragment lengths.

As illustrated in FIG. 43, all of these fragments are at intervals ofeither ˜34 residues or ˜17 residues which is the equivalent of a singletau repeat, or half of it. All of the fragments generated can thereforebe understood as arising from a simple set of proteolytic cleavagesoccurring at the positions indicated by the arrowheads in FIG. 43 from abasic heptameric aggregate, formed as shown in the figure. In thisscheme the fragments arise as the full combinatorial set of the proposedcleavages occurring at the 3 possible approximate positions shown by thearrowheads at either end of the aggregate. The corresponding predictedimmunoreactivity patterns seen with mAb 342 and T46 associated withthese fragments are also tabulated.

FIG. 44 shows these same fragments in descending order of length andincreasing gel mobility. Although the heptameric aggregate is shown forconvenience as arising entirely from full-length tau molecules, it willbe appreciated that the 12/14 kD fragment could be interposed within theproposed aggregate, replacing some of the binding partners, and that theprecise pattern of inclusion of these short fragments in the aggregatewill determine which precise fragments from the full set predominate ina given instance. Therefore, the production of this family ofproteolytic fragments is better understood as a possible repertoirewhich can be instantiated in various ways within the cell.

Example 4 Inhibitory Effects of Compounds on Production of ProteolyticFragment

Having achieved a stable cell system in which production of the 12 kDfragment (and others) could be controlled, the model was used to testthe inhibitory effects of reduced thionine. This is shown in FIG. 23. Ineach set of lanes, there is inducible production of the 12 kD band inthe presence of increasing concentrations of IPTG inducing higher levelsof T40. As the thionine concentration is increased, the production ofthe 12 kD band from T40 is suppressed. This is shown quantitatively inFIG. 24. In the absence of thionine, induction of T40 at increasingconcentrations of IPTG leads to a corresponding increased production ofthe 12 kD fragment. In the presence of 2 μM thionine, there is stillinduction of T40, but it is not converted into to the 12 kD fragment.

As reduced thionine is itself toxic, it is necessary to control forreduction in the levels of T40 induced by corresponding does of IPTG athigher levels of thionine. This can be achieved by determining the ratioof 12 kD:T40, which permits averaging the data across IPTG levels andshows a dose-dependent reduction in the level of the 12 kD relative tofull-length tau.

The activities of various compounds in the T40/12 kD assay are shown inFIGS. 9 to 16.

Results are expressed in terms of the ratio of 12 kD:T40 followinginduction of full-length tau (T40) by treatment cells with IPTG (0, 10,25, 50 μM) in the presence of thionine or tolonium chloride introducedat the concentrations shown in the presence of reducing agents (200 μMDTT/ascorbate), or chlorpromazine or tacrine introduced without reducingagents. As can be seen, thionine and tolonium chloride produceessentially identical inhibition, whereas chlorpromazine and tacrine arenon-inhibitory in the same concentration range. The effect of thereducing agents alone was tested in control experiments which showed nosignificant difference was seen in the 12 kD:T40 ratio in the presenceof reducing agents alone.

The properties of the cell line producing higher molecular weightdegradation products were also examined with MB and DMMB(dimethyl-methylene blue)

As can be seen in FIG. 45, DMMB proved to be surprisingly potent in thecell model. Its inhibitory activity could be seen both in the absence ofIPTG induction and following induction. Treatment with 1 μM DMMBeffectively abolished all degradation products within the cell. Furtherexperience with MB and DMMB has shown that even apparent base-lineproduction of the 12/14 kD species is largely determined by aggregation.That is, the constitutive production of the 295-391 fragment is itselfeither below the level of detection by immunoblot or else it isstabilised by spontaneous aggregation so as to reach levels within thecell which can be detected by immunoblot. Alternatively, the apparentbase-line levels of the 12/14 kD fragment seen without IPTG inductionand in the absence of treatment with a tau-aggregation inhibitor mayitself be dominated by templated aggregation-dependent production fromthe leakage levels of T40 produced in absence of induction. Whatever thecombination of factors which determines the levels of the 12/14 kDfragment in the base-line condition, its apparent expression can beessentially eliminated, along with higher molecular weight aggregationproducts, by a potent aggregation inhibitor such as DMMB. These resultsfurther confirm that production of the higher molecular weightproteolytic fragments (ie 30/32, 36/38, 42/44 kD) is also dependent oncritical tau-tau binding interactions occurring through the repeatdomain, as shown in FIGS. 41, 43 and 44.

FIG. 46 shows the activity of DMMB on base-line expression of the 12/14kD species, using the same set of assumptions regarding intracellulartau concentration and in vitro tau-tau binding affinity used in FIGS.10-16. In this case DMMB is found to have an apparent KI within the cellof 4.4 nM, and the cellular B50 value is ˜100 nM. This indicates thatDMMB is highly potent within the cellular milieu.

Example 5 Comparison of Inhibitory Effects of Reduced and OxidisedCompounds

The mathematical model used for the in vitro data was used to analysethe effects of test substances in the T40:12 kD cell assay. Using theknown values for Kd and KI from in vitro data, the expression indicatedwas used to solve for the intracellular concentration of full-length tau(see e.g. FIG. 10).

This was found to be about 500 nM, which is in the range expected fromstudies of tau in brain and in cell systems. A good fit to theexperimental data was obtained implying that for some compounds theinhibition of production of truncated tau within the cell can bepredicted from the approximate Kd and KI values determinedexperimentally in vitro.

Example 6 Examination of Inhibitory Properties of Diaminophenothiazines

In in vitro studies, the most active inhibitors of tau-tau bindingidentified were the reduced forms of diaminophenothiazines having 0, 2or 3 methyl groups. FIG. 25 shows the reduced forms of such compounds.The corresponding tau-tau binding curves are shown as a function ofmolar ratio with respect to tau in FIGS. 26 and 27. As shown, compoundsof the “desmethyl series” (0, 2 or 3 methyl groups) produceapproximately 50% inhibition of tau-tau binding (shown on the verticalaxis) at molar ratios of 3:1-4:1 of compound:tau ‘AMR’ shown on logscale on horizontal axis). The mean molar ratio for 50% inhibition oftau-tau binding for this group of compounds is 4:1.

Diaminophenothiazines having 4 or 6 methyl groups (the “methylatedgroup”) have a biphasic action, with enhancement of tau-tau binding atlower concentration, and inhibition of tau-tau binding at highconcentrations (FIG. 27). These compounds thus require much higher molarratios to effect 50% inhibition of tau-tau binding.

Examination of other features of the diaminophenothiazine compound wasalso carried out. Substitution of the heterocyclic nitrogen or sulphuratoms was found to severely interfere with inhibitory potency of thecompounds. Likewise, removal of the diamino groups was found to bedetrimental to the inhibitory activity. It thus appeared that thediamino and heterocyclic NB and S-structures are important for activityof the molecules in the inhibition of tau-tau binding.

For comparison, two methods were used to determine inhibitory activityin the tau-tau assay: STB is the mean tau-tau binding observed at 1 and10 μg/ml of compound at standard tau concentrations of 488 nM; LB50 islog10 molar ratio of compound:tau producing 50% inhibition of tau-taubinding (FIG. 28). As shown in FIG. 29, there is a strong correlationbetween the STB and LB50 values for a range of compounds, withchlorpromazine and riboflavin being two outliers (see also FIGS. 30 and31).

Example 7 Inhibitory Activity and Diffusion Potential

FIG. 32 indicates that there is a correlation between the number ofmethyl groups (NMETH) in a test compound and both the reductionpotential (E) and diffusion coefficient (DIF). In all comparisons, theSpearman rank correlation was used. As shown in FIG. 32, a stronginverse relationship between the number of methyl groups (NMETH) and thereduction potential can be seen only if methylene blue is excluded(normal type: correlation values including methylene blue; italic type:correlation values excluding methylene blue).

This indicates that methylene blue has a disproportionately highreduction potential relative to number of methyl groups (NMETH) in thisseries. There is also a strong positive correlation between the numberof methyl groups and the diffusion coefficient (DIF, FIG. 32).

As well as there being no observed correlation between the number ofmethyl groups and reduction potential (FIG. 33), it was surprisinglyfound that there was no observed correlation between reduction potentialand inhibitory potential (FIG. 34 b), although the extent of reductionof the diaminophenothiazines in the conditions of the assay is highlycorrelated with reduction potential (FIG. 33). And indeed, there is nocorrelation between the extent of reduction of these compounds andinhibitory potency (FIG. 34 a). On the other hand, there is a stronginverse correlation between the inhibitory potency of a compound and itsdiffusion coefficient, and it is possible to predict estimated LB50 andSTB values as linear functions of reduction coefficient and diffusioncoefficient when greater weighting is given to the diffusion coefficient(FIGS. 35, 36 and 37). Both the LB50 and STB values are found to beuniformly low for NMETH values up to and including 3, but for higherNMETH values (in particular methylene blue, NMETH=4) there is adisproportionately low inhibitory potency relative to the number ofmethyl groups. This may relate to the symmetric placement of the methylgroups which interferes with the stacking ability of the molecules, asmeasured by the diffusion coefficient. This can be seen, for example, inthe crystalline structure of methylene blue (see FIG. 38). Thediaminophenothiazine molecule is essentially flat and forms stackingarrays. The presence of charge in the molecule, as in the oxidised form,prevents the formation of such stacking arrays, and it appears to be thepropensity of the reduced form of this compound to form such stackingrelationships that determines the inhibitory potency of the series.

The experiments carried out by the present inventors examined thebinding of full-length tau in the aqueous-phase to the truncated repeatdomain fragment of tau in the solid-phase, as described in furtherdetail in WO96/30766. Binding was detected with either mAb 342 or mAb499. As shown in FIG. 39, there is typical tau concentration-dependenttau-tau binding in the presence of a large excess of the standardreducing agent dithiothreitol (DTT, 1 mM). However, the inhibitoryactivity of phenothiazines is also demonstrated in the presence of DTT(1 mM) in the standard configuration of the assay described above (i.e.the data for STB and LB50). The present inventors thus conclude that theinhibitory activity cannot be attributed to DTT per se, but rather tothe presence of the phenothiazines in their reduced form, due to anexcess of DTT.

In summary, the present inventors provide herein a potential,significantly improved, system for the treatment and prophylaxis ofdiseases such as Alzheimer's Disease in which proteins undergo inducedconformational polymerisation, e.g. as illustrated in the case ofAlzheimer's disease by pathological tau-tau binding. The importantteachings of this application, viz that the diffusion coefficient of acompound may important in determining its inhibitory potency towardsthis induced conformational protein polymerisation, are potentially ofgreat benefit in advancing our understanding of, and ability to providetherapy for, diseases such as Alzheimer's Disease. Finally, by combiningthe findings on the preferality of the reduced form of MB, anddemonstration of its activity in the cell-based assay at concentrationssubstantially below those predicted solely on the basis of in vitrodata, the inventors have shown that this compound, and others like it,could be used an appropriate reducing formulation for the prophylaxis ortreatment of AD and related disorders.

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1. A stable cell or cell line transfected with a vector comprising afirst nucleic acid encoding a tau protein and operably linked to aninducible promoter, and a second nucleic acid encoding a tau corefragment and operably linked to a constitutive promoter, wherein the taucore fragment is constitutively expressed at a non-toxic level in thecell or cell line, and wherein the tau protein is inducibly expressed inthe cell or cell line in response to a stimulus, and conformationalinteraction of the induced tau protein with the tau core fragment causespathological aggregation of the tau protein associated with a tauopathyand proteolytic processing of the tau protein into a tau productfragment comprising a tau core fragment.
 2. A stable cell or cell linetransfected with a vector comprising a first nucleic acid encoding amicrotubule-associated protein (MAP) and operably linked to an induciblepromoter, and a second nucleic acid encoding a tau core fragment andoperably linked to a constitutive promoter, wherein the tau corefragment is constitutively expressed at a non-toxic level in the cell orcell line, and wherein the MAP is inducibly expressed in the cell orcell line in response to a stimulus, and conformational interaction ofthe induced MAP with the tau core fragment causes pathologicalaggregation of the MAP associated with a tauopathy and proteolyticprocessing of the MAP into a product fragment comprising a corefragment.
 3. The stable cell or cell line of claim 2, wherein the MAP isMAP2.
 4. The stable cell or cell line of claim 1 or claim 2, wherein thetau core fragment extends from amino acid 295 to amino acid 391 of thefull-length tau protein and has SEQ ID NO:
 6. 5. The stable cell or cellline of claim 4, wherein the tau core fragment is a 12 kD paired helicalfilament (PHF)-core tau fragment.
 6. The stable cell or cell line ofclaim 1 or claim 2, wherein the tauopathy is a neurodegenerativedisorder and\or clinical dementia.
 7. The stable cell or cell line ofclaim 6, wherein the neurodegenerative disorder and\or clinical dementiaare selected from the group consisting of Alzheimer's disease, Pick'sdisease, Progressive Supranuclear Palsy (PSP), front-temporal dementia(FTD), parkinsonism linked to chromosome 17 (FTDP- 17),disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC),pallido-ponto-nigral degeneration (PPND), Guam-ALS syndrome,pallido-nigro-luysian degeneration (PNLD) and cortico-basal degeneration(CBD).
 8. The stable cell or cell line of claim 1 or claim 2, whereinthe cell or cell line is selected from the group consisting ofbacterial, mammalian, yeast and baculovirus cell or cell line.
 9. A kitcomprising the stable cell or cell line of claim 1 or claim 2 and anagent for stimulating production of the tau protein or MAP, or detectingthe interaction of the tau protein or MAP with the tau core fragment.10. The kit of claim 9, wherein the agent for detecting the interactionof the tau protein or MAP with the tau core fragment is an antibody. 11.The kit of claim 10, wherein the antibody is a monoclonal antibody whichis specific for a) a human-specific epitope located in the regionbetween Gly-16 and Gln-26 of tau; b) the core tau fragment truncated atGlu-391; c) a generic tau epitope in the repeat domain; or d) anon-species specific generic tau epitope located between Ser-208 andSer-238.
 12. A method for producing the stable cell or cell line ofclaim 1 comprising the step of transforming a cell with a vectorcomprising a first nucleic acid encoding a tau protein and operablylinked to an inducible promoter, and a second nucleic acid encoding atau core fragment and operably linked to a constitutive promoter, suchthat the tau core fragment is constitutively expressed at a non-toxiclevel in the cell or cell line, and the tau protein is induciblyexpressed in the cell or cell line in response to a stimulus.
 13. Amethod for producing the stable cell or cell line of claim 2 comprisingthe step of transforming a cell with a vector comprising a first nucleicacid encoding a MAP and operably linked to an inducible promoter, and asecond nucleic acid encoding a tau core fragment and operably linked toa constitutive promoter, such that the tau core fragment isconstitutively expressed at a non-toxic level in the cell or cell line,and the MAP is inducibly expressed in the cell or cell line in responseto a stimulus.
 14. A method for screening for a therapeutic agent forthe treatment of tauopathy comprising the steps of: (a) adding aputative therapeutic agent to the stable cell or cell line of claim 1 orclaim 2, (b) monitoring pathological aggregation of the induced tauprotein or MAP and/or proteolytic processing of the induced tau proteinor MAP into a product fragment comprising a core fragment in thepresence and absence of the putative therapeutic agent; and (c)identifying the therapeutic agent that blocks pathological aggregationof the induced tau protein or MAP by inhibiting proteolytic generationof the product fragment comprising the core fragment.
 15. The method ofclaim 14, wherein the proteolytic processing is monitored by monitoringthe production of a product fragment comprising a core fragment having amolecular weight selected from the group consisting of 12, 14, 25, 27,30, 32, 36, 38, 42 and 44 kD.
 16. The method of claim 15, wherein thecore fragment has a molecular weight of 12 kD.
 17. The method of claim15, wherein the production of the product fragment comprising the corefragment is monitored by SDS-PAGE.
 18. The method of claim 15 whereinthe production of the product fragment comprising the core fragment ismonitored immunologically.
 19. The method of claim 18 wherein theproduction of the product fragment comprising the core fragment ismonitored with a monoclonal antibody which is specific for a) ahuman-specific epitope located in the region between Gly-16 and Gln-26of tau; b) the core tau fragment truncated at Glu-391; c) a generic tauepitope in the repeat domain; or d) a non-species specific generic tauepitope located between Ser-208 and Ser-238.
 20. The method of claim 14,wherein the tauopathy is a neurodegenerative disorder and\or clinicaldementia.
 21. The method of claim 20, wherein the neurodegenerativedisorder and\or clinical dementia are selected from the group consistingof Alzheimer's disease, Pick's disease, Progressive Supranuclear Palsy(PSP), fronto-temporal dementia (FTD), parkinsonism linked to chromosome17 (FTDP-l 7), disinhibition-dementia-parkinsonism-amyotrophy complex(DDPAC), pallido-ponto-nigral degeneration (PPND), Guam-ALS syndrome,pallido-nigro-luysian degeneration (PNLD) and cortico-basal degeneration(CBD).